研究普立昂感染屏障與胺基酸序列的關係

୯ҥᆵ᡼εᏢғڮࣽᏢଣғϯࣽᏢࣴز܌

റγፕЎ

Graduate Institute of Biochemical Sciences College of Life Science

National Taiwan University Doctoral Dissertation

ࣴزදҥܳགࢉࡀምᆶữ୷ለׇӈޑᜢ߯!

Exploring the relationship of prion transmission barrier and amino acid sequence

ಷนࣔ

Chi-Chen Chuang

ࡰᏤ௲௤ȅഋٵⳮ റγ Advisor: Rita Pei-Yeh Chen, Ph.D.

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ύ๮҇୯102ԃ7Д July, 2013

ᖴᇞ!

!

!!!!᝽ӵࣁξǴ҂ԋ΋ᙀǴЗǴրЗΨǹ᝽ӵѳӦǴᗨᙟ΋ᙀǴ຾Ǵր۳ΨǶ!

ᅐߏΞٌधޑറγ੤ғఱಖܭ֋΋ࢤပǴགᖴऍ᜽ᆶඵችঋڀޑഋٵⳮԴৣ೭٤ ԃٰჹךޑऐЈࡰᏤᆶόܫకǴӢࣁாޑЍ࡭ωԖϞϺޑԋ݀Ǵாჹࣴزޑ዗

၈ǵԾߞаϷჹᏢғޑߥៈǴᡣךࡐቼ۩ૈၶـ೭ሶӳޑԴৣǶᗋाགᖴፓֽࢗ

୯ৎӼӄᆢៈೀᑵါ໚ೀߏǴԾவ຾Εፓֽࢗࡕா൩ჹךྣ៝ԖуǴᖴᖴாޑႴ ᓰᆶੵངǴΨᖴᖴாჹךޑߞЈǴᡣךԖ߿਻୲࡭ΠѐǴ׳གᖴாӧךϦ໒ᄽᖱ ᆶα၂่״ࡕಃ΋ਔ໔ᜢЈךǴᡣךߚதག୏ǶќѦགᖴЦയгԴৣǵ৪ᗱහԴ

ৣǵ໳Γ߾ԴৣаϷ׵ࡹ܃ԴৣӧԭԆϐύኘޜ߻ٰୖуךޑറγፕЎα၂Ǵ٠ ჹፕЎගрࡰ҅ᆶࡌ᝼Ǵᡣ೭ጇፕЎ׳ڀֹ᏾܄ǶΨགᖴፓֽࢗ᠘᛽ࣽᏢೀޑ܌

Ԗߏ۔ǴགᖴாॺޑЍ࡭ᡣך໩ճֹԋറγᏢՏǶ!

!!!!೭٤ԃٰǴӵ໯հНհཪԾޕǴགᖴჴᡍ࠻ޑԋ঩ჹךޑх৒ǴᖴᖴऱقᏢ ۂǴऐЈޑ㯍཮ךჴᡍ࠻ሺᏔޑᏹբמѯǵჴᡍλᄓ؁׳ᏼҺךޑЈᡫᏤৣǴ໒ Ꮴך೚ӭғࢲ΢གྷό೸ޑΓ٣ނǴҗ૱ޑᖴᖴیǶᗋाᖴᖴߞؼᏢߏǴᖴᖴா㯍

཮ךԖᜢϩ݋ኧᏵޑ೬ᡏᔈҔǴᡣךኧᏵ᏾౛ଆٰഢགᇸ᚞Ƕᖴᖴᇻӧऍ୯ޑ LilyᏢۂǴགᖴیԭԆϐύᗋाӣเךޑୢᚒǶགᖴ۸ᒑᏢ׌ǴନΑڐշךჴᡍ

ૈ໩ճ຾ՉѦᗋᔅךѺ౛ᅥ٣ǶགᖴਬണᏢ׌ǴଷВၟך΋ଆѐཚේ਻)གྷଆٗ

ࢤВη੿གྷပఽ*Ǵα၂ٗϺΨࡐԐ൩ډჴᡍ࠻ᔅךྗഢ΋ϪǴգ੿ࢂঁӳ࠸ηǶ གᖴླྀণᏢ׌ǴٌधգΑǴၟךӕ΋Ϻα၂܌аࡐӭ٣௃ΨளഞྠգᔅԆǶᖴᖴ

ඁ࣓ᏢۂᕴࢂࡐԖऐЈޑၟךಠϺǶᖴᖴ࢙׊Ꮲ׌㯍ך೚ӭቪፕЎޑλמѯǶᗋ ԖǴགᖴךӧፓֽࢗޑᏢߏॺǴᚶྷᏢߏǴா੿ࢂεӳΓǴӳЈޑၩךΠ੤ǵ᠋

ךᇥ٤πբ΢܈ғࢲ΢ޑελ٣ǴᖴᖴଭྭǴᖴᖴாӧךᄕًՐଣਔಃ΋ਔ໔ᇴ ډᙴଣྣ៝ךᗋວ१ނ๏ךکۂۂӞǴڬԴৣǴ΋ଆາ؁ޑ॥፪ᏢߏǴၟாາ؁

ޑਔ໔ᕴࢂอኩǵइమǵ຾᝼ǵЎܹǵሹໜ฻ᏢߏǴᖴᖴாॺྣ៝ךǴעך྽!

λл׌΋ኬǴӳӞӳވޑ٣ᕴ཮ऊך΋ଆୖᆶ)ךᄕًޑਔংգॺ΋ۓࡐᡋᓵ փ""*ǴᡣךޑଷВғࢲӭߍӭ࠮Ǵᖴᖴ҅ரᏢߏǴᖴᖴாჹךޑྣ៝Ǵᡣךӧ ౦ໂϝૈགڙډྕཪǶᖴᖴӕයӕᏢҏ୸Ǵהऐךޑᚯ๠਻)ᗨฅգࡐང࿗ۺ*Ǵ ٠ЪӧךനคշޑਔংഉӧךيᜐՔךࡋၸᜤᜢǶᗋाགᖴ᠘᛽ࣽᏢೀޑᏢߏۊ ॺǴᖴᖴாॺ೭ࢤਔ໔ޑх৒Ƕ!

!!!!നࡕךाགᖴךനᒃངޑৎΓ;ݿݿǵ༰༰ǵঢঢᆶۂۂǴӢࣁݿݿޑႴᓰ ךωൔԵፓֽࢗǴΨӢԜၶډ೭ሶӳޑߏ۔ᆶᏢߏॺǴᖴᖴ༰༰྽߃மॐךۺᆵ εǴךωૈၶډ೭ሶӳޑԴৣǴ਱Ǵգ΋ޔࢂךޑᄦኬǴך΋ޔ࣮๱գޑङቹ۳

߻຾)ՠࢂգёόёаόा؂ԛᜱךࡕǴᗋၟഞᇥךࡐಁۚฅ࣬ߞգޑ၉*Ǵךᒃ ངޑࠛࠛǴᗨฅךॺத֐ࢎǴՠךޕၰیࢂࣁךӳ)ۊۂࢂ֐όණᅀ*ǶவεᏢଆ ך൩΋ޔӧᆵч؃ᏢᆶπբǴӣৎޑਔংᕴࢂٰѐццǴᒱၸৎ္೚ӭελ٣ )kulo ۆۆჹόଆգ*ǴԶЪךΨததኪوǴՠգॺ҉ᇻЍ࡭ךǵႴᓰךǵഉՔךǴ ჹךคज़ޑх৒ᆶᡏፊǴᖴᖴǶΨᖴᖴ܌ԖᜢЈǵЍ࡭ךޑܻ϶ॺǶനനࡕךा

གᖴԴϺྭߥՙǴᡣךӧᆵчคፕࢂᏢ཰܈πբ೿΋Ϫ໩ճǴᖴᖴǶ!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ಷนࣔ!2013.07.25

I

ᄔा!

໺ኞ܄ੇᆟኬတੰࢂ΋ᅿठԝ܄ޑઓ࿶ଏϯ੯ੰ཮ቹៜ೚ӭথ٢ᜪ୏ނǴ

೷ԋԜੰޑচӢࢂҗߚ໺಍܄ޑགࢉੰচදҥܳȐprionȑ܌ЇଆǶPrion ޑ໺ኞ ӧӕނᅿ໔Кၨ৒ܰวғǴฅԶӧόӕނᅿϐ໔߾ڙज़ܭȨᅿ௼ࡀምȩǴΨ൩ࢂ

྽Ѧٰޑදҥܳữ୷ለׇӈऩࢂᆶஎЬޑữ୷ለׇӈ࣬՟ࡋৡ౦εޑ၉൩཮ቹ ៜჹ prion ޑ׎ԋ٠Ъό৒ܰၠᅿ໺ࢉǶࣁࣴزදҥܳགࢉࡀምᆶữ୷ለׇӈޑ ᜢ߯Ǵךॺᄒڗදၹᇡࣁදҥܳೈқനख़ाޑ΋ࢤữ୷ለׇӈ 108-144 ࣁኳࠠǴ ӝԋኧచදҥܳᴏ两Кၨځᜪᐘણ׎ԋޑ੝܄ǴԜѦ׳ճҔ඲ᅿᅀۓБݤໆϯр όӕނᅿ໔දҥܳ੯ੰޑགࢉࡀም٠ᔠෳόӕϐữ୷ለׇӈӵՖቹៜགࢉਏ౗Ƕ ჴᡍ่݀ᡉҢǴPrP ಃ 112 ဦූ୷ऩࣁҘ౷ữለǴ཮ߦ຾ᜪᐘણᠼᆢ׎ԋਔ ޑԋਡբҔǴऩಃ 139 ဦූ୷ࢂҘ౷ữለǵಃ 143 ဦූ୷ࣁํữለ߾཮ۯᒨԋਡ բҔǴՠऩӕ΋చᴏ两Ӹӧ΢ॊచҹਔǴᜪᐘણᠼᆢ׎ԋޑਏ݀཮೏ύکǶԜѦǴ ऩࢂаς࿶׎ԋᜪᐘણᠼᆢޑФȐbPrPȑࣁ඲ᅿ຾ՉЇ඲ჴᡍ཮ว౜Ǵಃ 129 ဦ

ූ୷ऩࢂқữለǵಃ 135 ဦූ୷ࣁϺߐоữለǵಃ 139 ဦූ୷ࢂҘ౷ữለǵಃ

143ဦූ୷ࣁํữለࣣ཮٬Ї඲ࡀምᡂଯǴՠऩ 138 ဦූ୷ࣁҘ౷ữለ߾཮फ़ե Ї඲ࡀምǴऩࢂቚуЇ඲ࡀምᆶफ़եЇ඲ࡀምٿޣቹៜӢηӕਔр౜ӧ΋చදҥ

ܳᴏ两΢Ǵ߾ٿޣޑቹៜ཮࣬ϕܢᎍǶ᏾ᡏԶقǴbPrP ჹܭ catPrPǵmPrP ᆶ bvPrP ឦܭեࡋЇ඲ࡀምǴჹܭ huPrPǵhaPrP ᆶ lionPrP ߾ឦܭύࡋЇ඲ࡀምǴԶჹܭ mdPrPǵdogPrP ᆶ pigPrP ឦܭଯࡋЇ඲ࡀምǶ

ऩࢂаς࿶׎ԋᜪᐘણᠼᆢޑчऍεفജȐmdPrPȑࣁ඲ᅿ຾ՉЇ඲ჴᡍǴ ჹbvPrPٰᇥឦܭեࡋᅿ௼ࡀምǴԶჹܭcatPrPǵlionPrPᆶmPrP߾ឦܭύࡋЇ඲

ࡀምǴԶჹܭbPrPǵpigPrPǵdogPrPǵhaPrPаϷhuPrP߾ឦܭଯࡋЇ඲ࡀምǶ

ᜢᗖຒǺදҥܳǵᜪᐘણᠼᆢǵЇ඲ǵᅿ௼ࡀምǵữ୷ለׇӈ

Abstract

Transmissible spongiform encephalopathies (TSE) are a group of fatal neurodegenerative diseases of mammals, caused by unconventional infectious agents known as “prion”. Transmission of prion between species is limited by a so called

“species barrier”. Prion formation could be affected dramatically if the amino acid sequences between the host PrP^C (cellular prion protein) and the inoculated prion are different, which results in the resistance of cross-species TSE transmission. In order to explore the correlation between the prion transmission barrier and the amino acid sequence, several prion peptides (sequences 108-144) were synthesizedΰthe sequence 108-144 based on the numbering of human prion protein sequenceα and the amyloidogenic properties of these peptides were compared. We used seeding titration method to quantify the seeding barrier and to examine how sequence difference affected the seeding efficiency. Our results showed that while residue 112 with methionine accelerated nucleation, residues 139 with methionine or 143 with serine retarded the nucleation process. But these effects could be neutralized when the substitutions with opposite effects had shown on the same peptide. Moreover, using the amyloid fibrils prepared from bPrP as seed, residues 129 with leucine, 135 with asparagine, 139 with methionine or 143 with serine substitution could increase seeding barrier, but residue 138 with methionine decreased seeding barrier. When both decreased and increased factors were appeared in the same peptide, their effects tended to cancel each other out. Overall, the relative seeding barrier is low for catPrP, mPrP, and bvPrP; moderate for huPrP, haPrP, and lionPrP; high for mdPrP, dogPrP, and pigPrP.

When the amyloid fibrils prepared from mdPrP were used as seed, the relative

III

seeding barriers for all these peptides were different from the bPrP seeding results: the relative seeding barrier is low for bvPrP; moderate for catPrP, lionPrP, and mPrP; high for bPrP, pigPrP, dogPrP, haPrP, and huPrP.

Keyword

Prion, amyloid fiber, seeding, species barrier, amino acid sequence

Ҟᒵ!

ᄔा ˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁI Abstract ... II Ҟᒵ ˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁˁIV კҞᒵ...VI ߄Ҟᒵ...VIII ᕭቪ߄ Abbreviations...IX

1.1 ೈқ፦ᒱᇤש᠄ᆶදҥܳ੯ੰ...1

1.2 ൂ΋ೈқᏢᇥȐprotein only hypothesisȑ ...6

1.3 දҥܳೈқ่ᄬ...7

1.4 ᜪᐘણᠼᆢޑ׎ԋ...11

1.5 දҥܳೈқϐᄬ׎ᙯᡂ...14

1.6 දҥܳϐၠނᅿ໺ࢉ...15

1.6.1 λႵᆶ௶ճ٥ঊႵ໔ޑᅿ௼ࡀም...17

1.6.2 ΓᜪᆶФϐ໔ޑᅿ௼ࡀም...19

1.6.3 Γᜪᆶജϐ໔ޑᅿ௼ࡀም...20

1.7 ځдނᅿϐᅿ௼ࡀምޑࣴز...22

1.8 ځдදҥܳᅿ௼ࡀምޑ࣬ᜢࣴز...22

1.9 ҁፕЎ୏ᐒ...23

ಃΒക ׷਑ᆶБݤ...25

2.1 ׷਑...25

2.1.1 Н...25

2.1.2 ϯᏢᛰࠔ...25

2.2 ሺᏔ...27

V

2.2.1 પϯǵϩ݋Ҕᆅࢊ...27

2.2.2 εࠠሺᏔ...27

2.3 Бݤ...28

2.3.1 දҥܳᴏ两ޑӝԋǵપϯᆶ᠘ۓ...28

2.3.2 а༝ΒՅӀ᛼इᒵදҥܳᴏ两ϐᜪᐘણᠼᆢ׎ԋ...36

2.3.3 аଯਏన࣬ቫ݋ሺ຾Չ዗ΚᏢྋှࡋޑϩ݋...42

2.3.4 аऀ೸Ԅႝηᡉ༾᜔ᢀෳදҥܳೈқᜪᐘણᠼᆢᙃ׎ᄊ...42

2.3.5 ಒझӸࢲᔠෳȐMTT cell viability assayȑ ...43

ಃΟക ่݀ᆶ૸ፕ...45

3. 1 ữ୷ለූ୷ӧᜪᐘણᠼᆢ׎ԋύ܌תᄽޑख़ाفՅ...45

3.2!!ނᅿ໔ᅿ௼ࡀምϷදҥܳ੯ੰၠᅿ໺ࢉޑຑ՗...54

3.2.1 Ї඲ჹᜪᐘણᠼᆢ׎ԋޑቹៜ...54

3.2.2 ᢀჸФȐbPrPȑᆶόӕނᅿ໔ϐᅿ௼ࡀም...55

3.3 ữ୷ለޑ่ᄬǵϯᏢ੝܄ᆶՏ࿼ჹදҥܳᴏ两ޑቹៜ...59

3.3.1 ஒ Met ࿼ඤԋ Val ܈ Leu...59

3.3.2 ஒ ValǵLeu ܈ Ile ࿼ඤԋ Met ...60

3.3.3 Serᆶ Asn ϕ࣬࿼ඤ...61

3.4 දҥܳᴏ两ޑ዗ΚᏢྋှࡋȐthermodynamic solubilityȑᢀჸ ...63

3.5 දҥܳᴏ两׎ԋϐᜪᐘણᠼᆢࠠᄊ...65

3.5.1 аऀ೸Ԅႝηᡉ༾᜔ᢀෳӚނᅿදҥܳᴏ两Тࢤׇӈ 108-144 .66 ᜪᐘણᠼᆢϐ׎ᄊ...66

3.6 ᢀჸчऍεفജȐmdPrPȑᆶόӕނᅿ໔ϐᅿ௼ࡀም...72

3.7 ᜪᐘણᠼᆢޑ߻០ނȐprecursorȑჹಒझࢥ܄ޑቹៜ ...77

ಃѤക ่݀ᆶ૸ፕ...82

ୖԵЎ᝘...86 ߕᒵ ΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞΞ98

!!!!!!!!!!!!!!!!კҞᒵ!

კ 1.1 অ҅ࡕϐೈқ፦ᄙ᠄ᆶ୴᠄ϐૈໆკ...2

კ 1.2 Ծ 1988 ԃԿ 2005 ԃ໔วғܭӄౚФଫϐ BSE ਢٯኧҞᆶΓᜪᑡ஻ vCJD ੰٯኧҞϐКၨკ...5

კ 1.3 දҥܳೈқϐ่ᄬ...9

კ 1.4 දҥܳҥܳ੯ੰวғਔ܌ౢғޑ PrP ᄬ׎ᙯඤ...10

კ 1.5 දҥܳೈқϐᜪᐘણᠼᆢ׎ԋኳࠠ...12

კ 1.6 ԋਡᆫӝኳࠠϐ੝ቻ...13

კ 1.7 දҥܳၠᅿ໺ࢉ...16

კ 2.1 ҁࣴزύ܌٬Ҕϐӝԋදҥܳᴏ两Тࢤׇӈ 108-144 ...29

კ 2.2 а MALDI-TOF MS ϩ݋ӝԋϐӚදҥܳᴏ两Тࢤ 108-144 ϐკ᛼ ...35

კ 2.3 ճҔ CD Ӏ᛼ᢀෳ bPrP ׎ԋᜪᐘણᠼᆢޑၸำ ...37

კ 2.4 ճҔ CD ᢀෳόӕނᅿϐදҥܳᴏ两ᜪᐘણᠼᆢ׎ԋϐғߏᒨᅉය ....39

კ 2.5 ϩձа bPrP ᆶ mdPrP ࣁ඲ᅿ຾ՉЇ඲...41

კ 2.6 ႝηᡉ༾᜔ۭΠᡉҢϯᏢӝԋϐΓᜪදҥܳᴏ两ȐhuPrPȑТࢤׇӈ..43

კ 2.7 MTT բҔᐒڋ ...43

კ 3.1 දҥܳᴏ两ӧԾฅ௃ݩΠ׎ԋᜪᐘણᠼᆢޑ௃׎...46

კ 3.2 ᆶ bPrP ࣬ৡ΋ঁữ୷ለϐނᅿǴځදҥܳᴏ两׎ԋᜪᐘણၸำϐ༝Βཱུ Ӏᇻ๋ѦӀӀ᛼...49

კ 3.3 ᆶ bPrP ࣬ৡΒঁữ୷ለϐނᅿǴځදҥܳᴏ两׎ԋᜪᐘણၸำϐ༝Βཱུ Ӏᇻ๋ѦӀӀ᛼...50

კ 3.4 ᆶ bPrP ࣬ৡΟঁữ୷ለϐނᅿǴځදҥܳᴏ两׎ԋᜪᐘણၸำϐ༝Βཱུ Ӏᇻ๋ѦӀӀ᛼...51

კ 3.5 ճҔ CD Ӏ᛼ᢀჸ bPrP ϐᜪᐘણᠼᆢჹӚදҥܳᴏ两ϐЇ඲բҔ ...57

კ 3.6 දҥܳᴏ两዗ΚᏢྋှࡋϐᢀჸ...64

VII

კ 3.7 Ӛނᅿදҥܳᴏ两Тࢤ 108-144 ׎ԋᜪᐘણᠼᆢӧ TEM Πϐ׎ᄊ...70 კ 3.8 ճҔ CD Ӏ᛼ᢀჸ mdPrP ϐᜪᐘણᠼᆢჹӚදҥܳᴏ两ϐЇ඲բҔ ...75

კ 3.9 ᆕᢀཥӝԋޑӭᴏ两ёૈޑၡ৩...78 კ 3.10 ϯᏢӝԋޑදҥܳᴏ两ჹಒझࢥ܄ޑቹៜ...80

კ 4.1 а bPrP ܈ mdPrP ࣁ඲ᅿჹӚނᅿදҥܳᴏ两ϐЇ඲ࡀም...85

߄Ҟᒵ!

߄ 1.1 Ҟ߻ࣁЗς೏ൔᏤϐ 16 ᅿදҥܳ੯ੰ...3

߄ 3.1 ᆶФදҥܳᴏ两Тࢤ࣬Кၨϐځдނᅿữ୷ለׇӈ...45

߄ 3.2 Кၨ mPrPǵhaPrPǵbvPrPǵbPrPrM109V ᆶ bPrPI139M ϐ 108-144 ữ୷ ለׇӈᆶғߏᒨᅉਔ໔...52

߄ 3.3 Кၨ catPrPǵlionPrP ᆶ mdPrP ϐ 108-144 ữ୷ለׇӈᆶғߏᒨᅉਔ໔ ...53

߄ 3.4 ϯᏢӝԋϐදҥܳᴏ两ϐғߏᒨᅉਔ໔...54

߄ 3.5 Кၨ MetǵValǵLeuǵIleǵAsn ᆶ Ser ϐ่ᄬᆶϯᏢ੝܄...62

߄ 3.6 КၨӚදҥܳᴏ两዗ΚᏢྋှࡋ...65 

߄ 3.7 දҥܳᴏ两׎ԋᜪᐘણᠼᆢࡕܭ TEM Πᢀჸ᏾౛ ...71

߄ 3.8 Кၨ CWD ёགࢉϐജဂϐදҥܳᴏ两 108-144 ୔໔ϐׇӈ...76

߄ 3.9 Ӛঁදҥܳᴏ两ځғߏᒨᅉයᆶჹಒझࢥ܄ޑቹៜ...81

߄ 4.1 දҥܳᴏ两ځԾว܄׎ԋᜪᐘણ୏ΚᏢǵ዗ΚᏢྋှࡋаϷЇ඲ࡀም84

IX

ᕭቪ߄ Abbreviation s

bPrP chemically synthesized bovine prion peptide ,sequence 108-144 BSE bovine spongiform encephalopathy

bvPrP chemically synthesized bank vole prion peptide,sequence108-144 CD circular dichroism

CJD Creutzfeldt-Jakob disease CWD Chronic wasting disease DCM dichloromethane

DMF N,N-Dimethylformamide

DMEM Dulbecco’s modified Eagle’s medium EDT 1,2-ethanedithiol

FBS fetal bovine serum Fmoc 9-Fluorenylmethoxycarbonyl GPI glycosyl phosphaidylinositol GSS Gerstmann-Straussler-Scheinker

haPrP chemically synthesized hamster prion peptide,sequence108-144

HPLC high performance liquid chromatography

huPrP chemically synthesized human prion peptide,sequence108-144 MALDI matrix-assisted laser desorption ionization

mdPrP chemically synthesized mule deer prion peptide,sequence108-144 mPrP chemically synthesized mouse prion peptide,sequence108-144 MTBE t-Butyl methyl ether

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

NaOAc sodium acetate NMM N-methylmorpholine NMR nuclear magnetic resonance PK proteinase K

PrP prion protein/peptide PrP27-30 N-terminally truncated PrP^Sc PrP^C cellular prion protein

PrP-res protease resistant form of prion protein PrP^Sc disease-causing isoform of prion protein PrP-sen protease sensitive form of prion protein

PyBOP benzotriazole-1-yl-oxy-tris-pyrrolidino-phosohonium hexafluorophosphate

rPrP recombinant prion protein

sCJD sporadic Creutzfeldt-Jakob disease SDS sodium dodecyl sulfate

TEM transmission electron microscope TFA trifluoroacetic acid TFE trifluoroethanol

TSE transmissible spongiform encephalopathy

ThT Thioflavin T

TIS Triisopropylsilane UV ultraviolet

vCJD variant Creutzfeldt-Jakob disease A (Ala) alanine

C (Cys) cysteine D (Asp) aspartic acid E (Glu) glutamic acid F (Phe) phenylalanine

XI G (Gly) glycine

H (His) histidine I (Ile) isoleucine K (Lys) lysine L (Leu) leucine M (Met) methionine N (Asn) asparagine P (Pro) proline R (Arg) arginine S (Ser) serine T (Thr) threonine V (Val) valine W (Trp) tryptophan Y (Tyr) tyrosine

ಃ΋കʳ ᆣፕ!

!

1.1 ೈқ፦ᒱᇤש᠄ᆶදҥܳ੯ੰ

ਥᏵϩηғނᏢޑਡЈ௲చǴೈқ፦ޑΟભ่ᄬȐtertiary structureȑࢂҗ΋

ભ่ᄬȐprimary structureǴջữ୷ለׇӈȑ܌،ۓǶೈқ፦ᄙ᠄Ȑfoldingȑၸำ ύԖ٤ҁيڀԖёԾՉᄙ᠄ޑૈΚǴԖ٤߾ሡाՔᒿೈқȐchaperoneȑޑڐշ ωૈֹԋᄙ᠄Ǵ೭٤Քᒿೈқёаᔅշೈқ፦Ԗਏ౗Ӧᄙ᠄ՠࠅό،ۓೈқ፦ޑ Ծฅ่ᄬȐnative structureȑǴᄙ᠄ֹԋࡕڀԖфૈޑೈқ፦ځ่ᄬߡࣁԾฅ่

ᄬǶȨenergy landscapeȩ ࢂҔٰඔॊೈқ፦ޑԾҗૈکځᄬ׎ϐ໔ޑᜢ߯Ǵ΋

૓ᇡࣁೈқ፦ޑԾฅ่ᄬջࢂѬॺനեૈໆϐᛙۓރᄊǴΨ൩ࢂӧfunnel shape നۭ೽ǴฅԶೈқ፦ᄙ᠄ޑၸำѸฅౢғ೚ӭᒱᇤᄙ᠄ȐmisfoldingȑϷ೽ϩᄙ

᠄ޑύ໔ނȐintermediateȑǴ೭٤ᒱᇤᄙ᠄Ϸ೽ϩᄙ᠄ύ໔ނёૈᆫ໣Զ׎ԋჲ ᆫ ӝ ނ (oligomers) Ǵ ӵ ݀ ೭ ٤ ჲ ᆫ ӝ ނ ؒ Ԗ ೏ ϩ ှ Ѭ ॺ ஒ ׎ ԋ ୷ চ ᠼ ᆢ Ȑprotofibrilsȑ٠࡭ុ୴᠄ᆫ໣ȐaggregatesȑǴ຾Զ׎ԋᜪᐘણೈқᠼᆢ(amyloid fibrils)Ƕаૈໆᢀᗺٰ࣮Ǵᜪᐘણᠼᆢ่ᄬڀԖଯࡋᛙۓ܄Ǵ΋ѿ׎ԋ߾όܰ೏

ઇᚯ܈ྋှΔԜ౜ຝᆶenergy landscape౛ፕ࣬ၴǴӢԜӧཥޑೈқ፦ᄙ᠄ૈໆኳ

ࠠύуΕΑೈқ፦ᄙ᠄ǵᆫ໣฻ፄᚇૈໆკǴԶᜪᐘણᠼᆢ߾֤ပӧКԾฅ่ᄬ

ૈໆ׳եޑૈໆᗺ΢Ȑკ1.1ȑ^{1; 2; 3; 4}Ƕᜪᐘણೈқ፦ԖόӕޑٰྍǴฅԶѬॺޑ ᆫ໣ᡏࠅᏱԖӅӕޑ੝ቻ⁵Ǻᜪᐘણೈқޑ؈ᑈނёаখ݀आȐcongo redȑࢉՅǴ ӧୃཱུӀȐpolarized lightȑྣ৔Π཮և౜᝴݀ᆘޑᚈש৔౜ຝȐbirefringenceȑǶ ԜѦᜪᐘણೈқᠼᆢӧx-rayޑྣ৔Π߾և౜ “cross-” ޑණ৔კኬǴᡉҢ

“cross-” ่ᄬёૈࢂᜪᐘણೈқᠼᆢޑӅ೯่ᄬǶ

ೈқ፦ᄙ᠄ၸำ཮ڙډѦࣚᘋ୏Զ೷ԋᒱᇤᄙ᠄ȐmisfoldingȑǴᏤठೈқ

፦ޑᆫ໣Ƕᆫ໣ϐೈқ፦཮ӧಒझಔᙃύౢғಔᙃ୴ᑈȐtissue depositionȑ٠а

ाхࡴߓ૟ੇᓨМੱ Ȑalzheimer’s diseaseǴADȑǵۦߎහМੱȐparkinson’s diseaseǴPDȑϷදҥܳ੯ੰȐprion diseaseȑ⁶Ƕ೭΋ᜪҗೈқ፦ᄬ׎ᡂ౦܌Їว ޑ੯ੰ΋૓ᆀࣁೈқ፦ᄬ׎ᡂ౦੯ੰȐconformational diseasesȑǴӵ݀Քᒿ๱ᜪ ᐘણೈқᠼᆢޑр౜Ψᆀࣁᜪᐘણೈқ੯ੰ Ȑamyloid diseasesȑǶ

კ1.1 অ҅ࡕϐೈқ፦ᄙ᠄ᆶ୴᠄ϐૈໆკ⁷Ƕቨࡋ߄Ң⪖(entropy)Ǵుࡋ߄Ң

ૈໆǴᜪᐘણᠼᆢ֤ပܭૈໆϐനեᗺǶ

දҥܳ੯ੰȐprion diseaseȑΞᆀ໺ࢉ܄ੇᆟރတੰȐtransmissible spongiform encephalopathyǹTSEȑࢂ΋ᅿठԝ܄ޑઓ࿶ଏϯ੯ੰȐneurodegenerative diseaseȑ

ΨࢂΓᆶ୏ނӅ೯ޑ੯ੰǴӢځ཮೷ԋΓᜪ܈୏ނတಔᙃੇᆟރੰᡂԶ೏ڮӜǶ ԜᅿੰੱӧࠠᄊᏢᆶੰ౛ғ౛Ꮲ΢ޑ੝ቻکځдᅌ຾ԄတੰǴӵߓ૟ੇᓨМੱǵ

܈ࢂۦߎහМੱᜪ՟ǴǴ୤΋όӕޑࢂදҥܳ੯ੰࢂ཮໺ࢉޑ ^{8; 9}Ƕӧόӕথ٢ᜪ

୏ނ໔දҥܳ੯ੰڀԖόӕޑӜᆀȐ߄ 1.1ȑǴӵӧξԲᆶᆟԲ໔ޑԲཝ᝚ੱ

Ȑ scrapie ȑǹ ӧ Ф ي ΢ ว ౜ ޑ ࣁ Ф ੇ ᆟ ኬ တ ੰ ᡂ Ȑ bovine spongiform encephalopathyǴBSEȑΞᆀئФੱȐmad cow diseaseȑǹӧജᆶᗸജ໔ޑജᄌ܄

੃઻੯ੰȐchronic wasting diseaseǴCWDȑаϷวғӧΓᜪي΢ޑ৤ၘМੱ

Ȑcreutzfeldt-Jakob diseaseǴCJDȑǵ৤ᎹੱȐkuruȑǵठڮ܄ৎ௼Ѩ઀ੱȐfatal familial insonmniaǴ FFI ȑ ک ਱ ථ ੝ ୗ М ᆕ ӝ ੱ ং ဂ Ȑ gerstmann-sträussler-scheinker syndromeǴGSSȑ฻Ƕ೭٤දҥܳ੯ੰ஻ޣޑεတѦ߄࣮ଆٰั༾๺ᕭǴεတಔ ᙃϪТӧᡉ༾᜔Πёᢀჸډတ೽և౜ੇᆟރ૓ޑޜࢰҭว౜ઓ࿶ಒझεໆԝ Ϋǵࢃރጤ፦ಒझቚғȐastrogliosisȑаϷڀԖೈқ䁙ܢלΚȐprotease-resistantȑ ޑදҥܳೈқ PrP^Scӧတ೽ಕᑈ׎ԋᜪᐘણᠼᆢȐamyloid-like fiberȑϷᜪᐘણඬ Ȑamyloid plaquesȑ฻ੰቻ¹⁰Ƕ

!

߄ 1.1 Ҟ߻ࣁЗς೏ൔᏤϐ 16 ᅿදҥܳ੯ੰǶ߄ӈࣁ੯ੰӜᆀᆶགࢉ୏ނᅿᜪ

ᆶ२ԛൔ֋ਔ໔¹¹Ƕ

നԐӧ1730ԃ൩ς࿶ԖԲཝ᝚ੱޑइᒵǴ྽ਔࢂӧम୯ᆟԲي΢ว౜ǴԜੰ

നεޑ੝ቻ൩ࢂวੰޑԲଫ཮يᡏว᝚όᘐޑᑃ㉵ᕅᏛǵᐋ༸ǵ᡽ડӢԶளӜǴ ԜѦੰԲᗋ཮և౜؁ቬόᛙǵ᠞םǵይ౳ǵᄕॹǵനಖԝΫ฻ੱރǶ྽ਔჹܭԲ ཝ᝚ੱޑੰচ٠όమཱǴฅԶӢࣁӧύኻࢬ໺ςΦ܌а྽ӦΓ࣬ߞԲཝ᝚ੱࢂ΋

ᅿᒪ໺܄੯ੰ٠ό཮໺ࢉ๏ΓᜪǴޔډ1930ԃѰѓǴݤ୯ᛌᙴ᛾ჴԲཝ᝚ੱࢂ΋

ᅿ໺ࢉੰǴԲཝ᝚ੱёаவᆟԲ໺ࢉډξԲǴӆவξԲ໺ࢉ๏ᆟԲǶԲཝ᝚ੱന εޑੰቻ൩ࢂတ೽р౜ޜݰЪࢃރઓ࿶ጤಒझԖό҅தቚғޑ౜ຝǹԶӧ1950 ԃܭЃѲ٥ཥ൳ϣ٥р౜Α΋ᅿћ଺Ȩ৤Ꮉȩޑ੯ੰǴGajdusekϷځдࣴزޣӧ

஻ޣޑᓐ೽ว౜ԖᐘણኬೈқඬȐamyloidȑӸӧǴவ஻ޣғࢲಞᄍύளޕдॺ Ԗ१ҔԝѐᒃΓࡁᡏޑಞ߫ǴӢԜ௢ෳࢉੰচӢᆶԜԖᜢǴԜѦКၨԲཝ᝚ੱᆶ

৤Ꮉੱ஻ޣว౜ٿޣੰ౛࣬՟Ǵᗨฅ҂ϩᚆрҺՖੰচᡏǴՠ᛾ჴ৤Ꮉੱҭࢂ΋

ᅿ໺ࢉ܄੯ੰǶวғӧΓᜪനԐޑදҥܳ੯ੰࢂӧ1920ԃҗCreutzfeldtکJakobϩ ձว౜ޑੰੱǴੰว߃ය૶ᏫΚ཮૰ଏǵ؁ቬόᛙ฻ᜪ՟࿆քޑੱރǴᒿ๱ੰำ

຾৖Ǵ஻ޣޑѤަᆶ᙮༸཮όଶޑቃਗ਼ܜ୏Ǵԝࡕှওࡁᡏว౜஻ޣတ೽ᝄख़ڙ ཞࠅؒԖวݹޑ௃ݩǴԶ೭ᅿੰ൩೏ڮӜࣁ৤ၘМੱǶ৤ၘМੱޑੰӢԖΟᅿኳ ԄǺȐ1ȑଽว܄ȐsporadicǴ~85ʘȑǺਥᏵЎ᝘Ǵଽว܄ޑ৤ၘМੱȐsporadic CJDǴ sCJDȑวғ౗ऊԭ࿤ϩϐ΃Ǵੰٯ႟ࢃр౜ӧӄౚӚӦǴੰӢҞ߻όܴǴεӭ ኧ஻ޣวੰԃសቫቶݱǴੰำឦ٥࡚܄ȐsubacuteȑǴ஻ޣ߃යཀדόమԶࡕᄽᡂ ԋѨඵȐdementiaȑǵၮ୏ઓ࿶ѨፓȐataxiaȑ฻ੰቻǴ຾ำِೲǴᝄख़ޣதܭว

ੰࡕ1ԃϣԝΫǴੰ౛ϪТว౜஻ޣନλတ೏ᝄख़ઇᚯѦǴεတҜ፦ೀҭԖੇᆟ ኬޑޜࢰǹȐ2ȑᒪ໺܄ȐinheritedǴ~15%ȑǺᒪ໺܄ޑ৤ၘМੱȐfamilial CJDǴ fCJDȑࣁৎ௼܄ᡉ܄ᒪ໺ǴЬाࢂӢࣁදҥܳೈқޑ୷ӢPRNPวғँᡂ܌೷ԋǴ ԜѦठڮ܄ৎ௼Ѩ઀ੱȐFFIȑک਱ථ੝ୗМᆕӝੱংဂȐGSSȑ฻ࣣឦܭᒪ໺

܄දҥܳ੯ੰǹȐ3ȑགࢉ܄ȐinfectiousǴɦ1%ȑǺΞᆀࣁᙴྍ܄৤ၘМੱȐiatrogenic

CJDǴiCJDȑЬाࢂӢࣁӚᅿᙴᕍՉࣁ܌೷ԋޑགࢉǴନڙԦࢉޑᙴᕍᏔ׷܈ቲ కނǵᛰނ฻གࢉѦǴനӭࢂӧݙ৔๧ڗٰԾΓᜪࡁᡏϐတΠࠟᡏ಻ᅟᆾࡕаϷ Ꮤ۔౽෌วੰޑঁਢǶ

ӧΒΜШइ҃ኻࢪᛈวޑئФੱǴ೏ᚶᅪᆶਔ໔࣬ৡόΦޑᡂ౦܄৤ၘМ

ੱȐvariant CJDǴvCJDȑԖᜢǹئФੱޑੰӢёૈࢂӢࣁᗯ१ФଫޑႩ਑္ᄞԖ Բཝ᝚ੱޑԲ଺ԋޑମણ܌ᏤठǴচӃႩ਑ᄞମણޑҞޑӧܭᡣФޔௗ֎ԏೈқ

፦ᡣФߏள׳֧Ǵ่݀೷ԋ BSE ᛈวޑࣝ௃Ƕᡂ౦܄৤ၘМੱȐvariant CJDǴ vCJDȑϐੰٯࢂӧ 1995 ԃ२ࡋӧम୯೏ว౜Ǵᆶၸ۳৤ၘМੱੰٯόӕೀӧܭǴ ཥᜪࠠޑ৤ၘМੱวੰԃសቫදၹୃեȐѳ֡ԃសऊ 28 ྃѰѓȑ¹²౒ෳ஻ޣё

ૈࢂҗܭ१Ҕ܈ௗ᝻ BSE ੰФϐಔᙃ܌Ꮴठ^{13; 14}Ƕ

!

!

კ1.2 Ծ1988ԃԿ2005ԃ໔วғܭӄౚФଫϐBSEਢٯኧҞᆶΓᜪᑡ஻vCJDੰ

ٯኧҞϐКၨკ¹⁴Ƕ

ئജੱȐCWDȑҭࢂ΋ᅿё໺ኞޑੇᆟኬတੰǴҁੰ࿶ԾฅགࢉޑജࣽԖ ࢶᕚξᗸജȐrocky mountain elkȑǵчऍεفജȐmule deerȑǵқ׀ജȐwhite-tailed deerȑаϷ໵׀ജ฻Ǵቹៜ܌Ϸό໻ࢂഁӦജଫǴࣗԿࢂΓᜪ੬Ꭶޑᒙᜪ୏ނҭ ว౜Ԗᄌ܄੃઻܄ੰޑӸӧǶനԐӧ1967ԃܭऍ୯Ջ೽ว౜Ǵ΋ଫчऍεفജр

౜੃઻܄ੱংဂǴ੝ቻࢂᡏख़ᅌᅌ෧ᇸനࡕ૰১ठԝǴޔډ1978ԃω೏ዴᇡࣁ໺

ኞ܄ੇᆟރတੰ٠Ъς࿶ණኞډчऍǵу৾εǵቺ୯ᆶࠄᗬ฻Ӧ^{15; 16; 17}ǶCWD εӭวғӧԋԃ୏ނǴᖏ׉ੱރࣁᡏख़όᘐ෧Ͽǵᆶӕឯ໔ޑ୏ނϕ୏෧Ͽǵࢬ తǵ໯Нໆᆶ௨ֿໆᗊቚ฻Ǵᗸജ߾ԖၸࡋᑫᏟϷઓ࿶፦฻ੱރǴᗨฅ཮༐Ӟዼ ᜪՠჹ૛਑ޑᑫ፪εεफ़ե^{18; 19}ǶCWDޑ໺ኞኳԄ٠όమཱՠவࢬՉੰᏢفࡋᢀ ჸ܈೚ࠟޔགࢉϷНѳགࢉࣣԖёૈǴᗨฅCWD৒ܰӧജࣽ୏ނ໔໺ኞՠჹᆶ ϐӕۚޑФǵᆟԲǵξԲ฻୏ނࠅό཮р౜CWD܈ࢂTSEϐੱރǴԜѦགࢉྍ

ࢂցٰԾൄనǵֿన܈ᕨߡԦࢉϐ१ҔН܈Ⴉ਑٠คޔௗ᛾Ᏽ²⁰ǹᗨฅҞ߻٠ؒ

ԖCWDੰচ཮໺ࢉ๏Γޑ᛾ᏵǴՠදҥܳ੯ੰወҷයࡐߏǴ१ҔജԺ܈ᗸജԺ ёૈሡ࿶ၸࡐߏਔ໔ω཮ठੰ²¹Ǵऩ੿ԖԝܭԜᅿ໺ࢉੰޑ஻ޣࡐԖёૈ೏ᘜᜪ ࣁόܴচӢǴԶό཮ᖄགྷᆶئജੱԖᜢǴ܌аҞ߻ό௨ନ཮ߟ্ΓᡏǶ

1.2 ൂ΋ೈқᏢᇥȐprotein only hypothesisȑ

1982ԃǴPrusiner S.BჹЇଆԲཝ᝚ੰޑགࢉੰচΠΑ΋ঁۓကǺȨprionȐύ

᝿ࣁදҥܳȑࢂ΋ᅿό֖ਡለޑೈқ፦གࢉಈηȐproteinaceous infectious particles without nucleic acidȑȩ²²ǶPrusinerϐ܌аගрԜᇥݤࢂҗܭന߃΋໒ۈჹܭᑡ஻

Բཝ᝚ੰޑੰচ٠όమཱǴ྽ਔޑᏢޣࡰᆀࢂҗᄌ܄ੰࢥȐslow virusȑ܈ࢂߚ

໺಍܄ੰࢥȐnonconventional virusȑ܌Ꮴठϐ໺ࢉੰ^{23; 24}ǴฅԶAlper฻Γว౜Բ ཝ᝚ੰޑੰচჹܭႝᚆᒟ৔ᆶ๋Ѧጕ฻ڀԖઇᚯਡለૈΚޑଯૈໆ৔ૈڀԖܢ

לΚ²³ǴӢԶЇวᏢೌࣚჹܭЇଆԲཝ᝚ੰޑੰচ዗ਗ਼૸ፕǶੰচЬाԋϩᇥݤ ϩݔǴԖޑଷ೛ࢂλࠠDNAੰࢥȐsmall DNA virusȑǵӭᗐᡏ܈ࢂೈқ፦฻⁹Ǵฅ Զӧ1967ԃGrififithගрԲཝ᝚ੰޑੰচёૈࢂࢌᅿόሡा࿶ၸᒪ໺ނ፦Ȑٯӵ DNA܈RNAȑޑፄᇙᆶᘉණջڀԖԾךፄᇙૈΚȐself replicatingȑޑೈқ፦²⁵Ǵ ӢԜԖΑȨൂ΋ೈқ፦ᏢᇥȐprotein only hypothesisȑȩౢғǴ1982ԃBoltonவڙ ډԲཝ᝚ੱགࢉޑ௶ճ٥ঊႵȐsyrian hamsterȑޑတಔᙃύϩᚆр΋ᅿჹೈқ䁙 KȐprotease KȑԖܢל܄ȐresistanceȑЪϩηໆϟܭ27~30 kDaϐೈқ፦²⁶Ǵ᛾ჴ දҥܳೈқޑЬाԋϩࣁԜǴϐࡕࣴزว౜Ǵ࿶ၸપϯளډޑPrPڀԖԲཝ᝚ੱ

ޑགࢉΚԶЪځགࢉΚёᙖҗࢌ٤ઇᚯೈқ፦ޑೀ౛܈ࢂ٬ҔPrPלᡏ٬ϐ෧১

26; 27

ǹԶӧPrPޑ୷ӢPRNP೏ว౜аࡕǴว౜Ԝ୷Ӣҁٰ൩ӸӧܭΓᜪᗋԖ೚ӭ

୏ނޑࢉՅᡏύǴԶ೷ԋදҥܳೈқठڮޑӢનӧܭPRNP୷Ӣౢғँᡂ^{10; 28}Ǵ ऩࢂεໆ߄౜PRNPँᡂޑᙯ෗λႵǴ߾λႵ܌ౢғޑё໺ࢉ܄ઓ࿶ଏϯ੯ੰੱ

ރکᑡ஻දҥܳ੯ੰᜪ՟²⁹ǴԜѦऩࢂPRNP୷ӢকନȐPRNP^-/- knocked out miceȑ ޑλႵ߾ჹԲཝ᝚ੱڀԖܢלΚ³⁰Ǵ೭٤ჴᡍ่ࣣ݀ࢂЍ࡭Prusinerޑൂ΋ೈқᏢ ᇥǶ

1.3 දҥܳೈқ่ᄬ

දҥܳೈқȐprion proteinǴPrPȑࢂҗPRNP୷Ӣ܌ᙯ᝿Ǵᙯ᝿ࡕޑPrP֖Ԗ 254ঁữ୷ለූ୷Ȑamino acid residueȑǴځữ୷ᆄȐN-terminalȑᆶ♐୷ᆄ ȐC-terminalȑӚ֖Ԗ΋ঁऊ20ঁữ୷ለූ୷ޑߞဦᴏ两Ȑsignal peptideȑǴӧᙯ ᒵၸำύߞဦᴏ两཮೏Ϫନฅࡕԋࣁ֖Ԗ209ঁữ୷ለූ୷Ȑׇӈ23-231ȑϐԋ ዕޑPrPǴԋዕޑPrPࢂ΋ᅿૈکಒझጢ΢ϐGPIȐglycosylphosphatidylionsitolȑౢ

ғӅሽᗖ่ޑೈқ፦ȐGPI-anchoredȑǴPrPճҔځSer231ᆶGPIϩη่ӝ٠ߕ๱ܭ ಒझጢѦୁ³¹ǴԜѦPrPӧCys179ᆶCys214໔֖Ԗ΋ᚈ౷ᗖȐdisulfide bondȑǴԶ

ׇӈ΢ϐAsn181ᆶAsn197ૈᆶේ᜘ᗖ่ౢғᗐϯբҔȐN-linked glycosylationȑ

Ȑკ1.3Aȑ³²Ƕ

PrPޑữ୷ᆄ֖Ԗ5ঁ8ख़ፄޑᴏ两Ȑoctapeptide repeatsȑǴځфૈ௢ෳૈᆶል ᚆη่ӝ٠ፓ࿯ځࡡۓǵל਼ϯᓸΚǵಒझڰ๱аϷૻ৲໺ሀԖᜢ^{10; 33}ǶPrPӸ ӧܭӚނᅿ໔хࡴথ٢୏ނǵങᜪаϷചᜪ฻ǴځߥӺ܄ଯЪӧᡏϣ೚ӭಔᙃӵ Ј᠌ǵମᓝԼаϷ๝᠌ΨԖPrPӸӧǴځύаઓ࿶ಒझ߄౜ໆനଯ³⁴ǴճҔਡᅶ ӅਁȐnuclear magnetic resonanceǴNMRȑӀ᛼ςԋфှ݋ΓᜪȐhumanǴHomo sapiensȑǵ௶ճ٥ঊႵǵλႵȐmouseǴMus musculusȑаϷФ٥ࣽ୏ނȐbovineȑ

฻ނᅿϐӄߏPrPख़ಔೈқȐrecombinant proteinȑ35; 36; 37; 38

ǶаΓᜪPrPࣁٯǴNMR

ှрϐ่ᄬᡉҢځữ୷ᆄೀ൤ቸ܄Ǵ֖Ԗऊ100ঁữ୷ለූ୷҂ڀ੝ۓϐΒભ่

ᄬǴ♐ለᆄೀ߾ڀၨڰۓޑౚރ่ᄬȐglobularȑǴځύхࡴ3చhelixǵ2ިE-sheet аϷ1ჹೱௗಃΒచϷಃΟచD-helixޑᚈ౷ᗖȐკ1.3Bȑ³⁸Ƕ

ȐAȑ

ȐBȑ

კ1.3 දҥܳೈқϐ่ᄬǶȐAȑ҅தϐදҥܳೈқ፦΃ભ่ᄬǴׇӈ1-254ࣁ ᙯ ᝿ ࡕ ҂ ࿶ অ Ⴌ ϐ ߏ ࡋ Ǵ Ϫ ѐ ߻ ࡕ ޑ ߞ ဦ ᴏ 两 ߡ ё ԋ ࣁ ԋ ዕ ޑ PrP Ȑ ׇ ӈ 23-231ȑǴNᆄޑ5ख़ፄoctarepeatёکልᚆη่ӝǴCᆄ߾ࢂڀԖΒભ่ᄬȐ3 D-helixǵ2 E-sheetȑᆶ੝ਸঅႬȐᗐϯǵᚈ౷ᗖȑೀǶȐBȑΓᜪPrP23-230ख़ಔ

ೈқϐNMR่ᄬ³⁸Ǵữ୷ᆄࣁ໳Յ຀ጕೀǴᡉҢࣁׇӈ23-121ǴӢ൤Ԗቸ܄ӢԜ คݤှрዴϪ่ᄬǹ♐୷ᆄԖᡉҢ3చᐊՅޑD-helixȐׇӈ144-154ǵ173-194ǵ 200-228ȑǵᙔՅ2ި࣬ϕϸӛѳՉϐE-sheetȐׇӈ128-131ǵ161-164ȑаϷคڰ ۓ໳ՅჴጕϐΒભ่ᄬǶ

දҥܳೈқϩηໆऊӧ33-35 kDa٠ЪӸӧٿᅿ่ᄬ౦ᄬނȐisoformȑȐკ 1.4ȑǺ΋ᅿࢂ҅தᄬ׎ޑPrP^CȐC߄ҢcellularȑǴќ΋ᅿ߾ࢂᆶTSE࣬ᜢޑᄬ׎

PrP^ScȐSc߄ҢscrapieȑǴ྽දҥܳ੯ੰวғਔǴදҥܳೈқ่ᄬ཮җPrP^Cᙯᡂԋ PrP^ScǶ೷ԋٿᅿPrPᄬ׎ӸӧޑচӢԖٿᅿଷ೛Ǻ΋ࢂᇡࣁҗܭᙯ᝿ࡕঅႬբҔ Ȑpost-translational modificationȑ܌ᏤठǴՠനࡕࠅؒԖளډҺՖ่݀³⁹ǹќ΋ᅿ

ଷ೛߾ᇡࣁPrP໻วғᄬ׎΢ϐׯᡂ٠คҺՖׇӈ܈ᙯ᝿ϐঅႬ²⁵ǴฅԶԜཷۺ ࠅᆶೈқ፦ࡌᄬ౛ፕȨone sequenceǴone structureȩ࣬ၴङ⁴⁰ǶӕׇӈޑPrP^Cᆶ PrP^Scό ໻ ӧ ่ ᄬ ΢ Ԗ ܌ ৡ ౦ Ǵ ځ ғ ϯ ੝ ܄ ᆶ ठ ੰ Κ ҭ Ԗ ܌ ό ӕ Ǻ ྽ ว ғ PrP^CPrP^Scᄬ׎ׯᡂਔǴځΒભ่ᄬ֖ໆวғׯᡂǴPrPচҁаD-helixࣁЬᙯᡂ ԋаE-sheetࣁЬ³⁹ǴԜѦPrP^Cૈ೏ೈқ䁙KНှǵૈྋܭߚᚆη܄మዅᏊЪόڀ

׎ԋᜪᐘણᠼᆢޑ໼ӛǹԶPrP^Sc߾ჹೈқ䁙KڀԖ೽ҽܢלΚȐεऊ142ঁữ୷ ለූ୷Ǵϩηໆ27-30 kDaѰѓȑǵคݤྋှܭߚᚆη܄మዅᏊՠڀԖ׎ԋᜪᐘ ણᠼᆢޑ໼ӛ^{9; 41}Ƕ

კ1.4 දҥܳҥܳ੯ੰวғਔ܌ౢғޑPrPᄬ׎ᙯඤǶচҁᄬ׎҅தϐPrP^C ȐD-helixᆶE-sheetޑ֖ໆϩձࣁ40%ᆶ3%ȑᙯඤԋᄬ׎ό҅தЪڀགࢉΚϐ PrP^ScȐკύ่ᄬࣁ௢ෳ่ᄬ, D-helixᆶE-sheetޑ֖ໆϩձࣁ30%ᆶ45%ȑǶ

1.4 ᜪᐘણᠼᆢޑ׎ԋ

Prusinerᇡࣁ྽PrP^Cᙯᡂ׎ԋPrP^ScਔǴᄬ׎ό҅தޑPrP^ScёȨགࢉȩတ೽ϣ

଼நޑPrP^C೷ԋځ่ᄬޑׯᡂǴ٬ޑ่ᄬ౦தޑPrP^Scӧတ೽ᆫ໣׎ԋᜪᐘણᠼᆢ

຾Զቹៜ҅தޑတ೽фૈǴനࡕ཮׎ԋႽੇᆟᡏރޑ่ᄬǶᏃᆅჹܭᄬ׎ᙯඤޑ ᐒڋ٠όమཱǴՠҞ߻ЬाԖٿᅿᜪᐘણᠼᆢ׎ԋϐኳࠠၨቶݱ೏ௗڙǺኳ݈Ї Ꮴ ӆ ש ᠄ ኳ ࠠ Ȑ template-directed refolding model ȑ ᆶ ԋ ਡ ᆫ ӝ ኳ ࠠ Ȑnucleation-polymerization modelȑǺ

Ȑ1ȑ ኳ݈ЇᏤӆש᠄ኳࠠ

ӧԜኳࠠύǴPrP^Scϩηޑ׎ԋόܰЪ጗ᄌǴӢࣁҗPrP^CᙯඤԋPrP^Scޑၸ ำύѸ໪ၠຫ࣬྽ଯޑૈໆࡀምȐenergy barrierȑǴӢԜǴԜၸำԋࣁᜪᐘણ׎

ԋϐೲ౗،ۓ؁ᡯȐrate-determining stepȑǴόၸऩࢂς࿶ԖPrP^ScࢂӸӧǴ߾ё

྽բኳ݈ڐշ٠уזPrP^Cᙯඤԋ่ᄬᛙۓޑPrP^ScǴ٬ᜪᐘણᠼᆢזೲۯߏޔډၲ

ѳᑽࣁЗȐკ1.5aȑǴᗨฅԜኳ࣮ࠠ՟Ԗ౛ՠ٠҂Ԗჴᡍ᛾ᏵᡉҢԖᛙۓޑPrP^Sc

ൂᡏȐmonomerȑӸӧ⁴²Ƕ

Ȑ2ȑ ԋਡᆫӝኳࠠ

PrP^Cᆶ PrP^Sc໔ ޑ ᙯ ඤ ೀ ܭ ዗ Κ Ꮲ ΢ ޑ ѳ ᑽ ރ ᄊ Ȑ thermodynamic equilibriumȑǴ٠Ъ໼ӛаPrP^Cᄬ׎ӸӧǴ዗ΚᏢ΢όᛙۓޑPrP^Scሡ۶Ԝ่ӝ׎

ԋ੝ۓελޑჲᆫᡏωૈᛙۓӸӧǴԶԜჲᆫᡏջࣁ׎ԋᜪᐘણᠼᆢਔ܌ᆀޑ඲

ਡȐnucleusȑǴҗܭ඲ਡ׎ԋόܰǴӢԜǴԜ؁ᡯࣁᜪᐘણᠼᆢ׎ԋਔϐೲ౗،

ۓ؁ᡯǶ྽඲ਡ׎ԋࡕǴPrP^Sc൩ёаԖਏޑబуӧ඲ਡ΢Զۯߏᜪᐘણᠼᆢޔ

ၲѳᑽࣁЗȐკ1.5bȑ⁴³Ƕ

კ 1.5 දҥܳೈқϐᜪᐘણᠼᆢ׎ԋኳࠠ⁴⁴ΖȐaȑኳ݈ЇᏤӆש᠄ኳࠠǺଷ೛

PrP^ScൂᡏК PrP^C׳ᛙۓǴՠჴሞ΢ࢂόёૈၲډޑǹPrP^Scൂᡏޑр౜ࢂଽว ޑ܈ࢂѦٰ܌ගٮǴѬёа྽բኳ݈ЇᏤځд҅தᄬ׎ϐ PrP^Cᙯᡂԋό҅தᄬ

׎ϐPrP^ScǶȐbȑԋਡᆫӝኳࠠǺ඲ਡޑ׎ԋࢂԜኳࠠޑೲ౗،ۓ؁ᡯǴ΋ѿ඲

ਡ׎ԋࡕߡёᛙۓӸӧǴԜਔPrP^Scൂᡏߡёזೲబуځ΢຾Զۯߏᜪᐘણᠼᆢ ޑߏࡋǶ

ӧ ԋ ਡ ᆫ ӝ ኳ ࠠ ύ Ǵ PrP^Scൂ ᡏ ޑ ᐚ ࡋ ሡ ଯ ܭ ܌ ᒏ ޑ ᖏ ࣚ ᐚ ࡋ Ȑ critical concentrationȑωૈ࣮ډᜪᐘણᠼᆢޑ׎ԋǴեܭᖏࣚᐚࡋЬाࢂаPrP^Scൂᡏࣁ ЬǴऩࢂೈқ፦ᐚࡋᇻຬၸᖏࣚᐚࡋǴ߾ᜪᐘણᠼᆢᐚࡋ཮࡭ុቚуǴՠPrP^Sc

ൂᡏޑᐚࡋ߾ᆢ࡭όᡂȐკ1.6AȑǹԜѦᜪᐘણᠼᆢ׎ԋϩࣁΟ໘ࢤǴ٩ׇࣁ඲

ਡ׎ԋਔޑᒨᅉයȐlag phaseȑǵPrP^Scൂᡏזೲబуܭ඲ਡ΢ϐۯߏයȐelongation phaseȑаϷᜪᐘણᠼᆢᆶPrP^ScൂᡏೀܭѳᑽόӆቚߏޑѳᑽයȐequilibrium phaseȑȐკ1.6BȑǹऩᒨᅉයਔѦу඲ᅿȐseedȑ߾ᜪᐘણᠼᆢޑ׎ԋёҥջ໒

ۈȐკ1.6Cȑ⁴⁵Ƕ

ȐAȑ

ȐBȑ ȐCȑ

კ1.6 ԋਡᆫӝኳࠠϐ੝ቻ⁴⁵ȐAȑᜪᐘણᠼᆢޑ׎ԋࢂ٩ྣೈқ፦ᐚࡋ،ۓǴ PrP^ScൂᡏᐚࡋሡଯܭᖏࣚᐚࡋȐCRȑωૈ׎ԋᜪᐘણᠼᆢǹȐBȑᜪᐘણᠼᆢ׎

ԋ߻ࡕਔයࣁ጗ᄌޑԋਡϸᔈǴ΋ѿ඲ਡ׎ԋᜪᐘણᠼᆢߡёזೲԋߏޔၲѳ ᑽǴԜਔൂᡏޑᐚࡋ཮࡚ೲΠफ़ࣗԿեܭᖏࣚᐚࡋǹȐCȑѦу඲ᅿёа٬ᜪᐘ ણᠼᆢޑ׎ԋၢၸᒨᅉයԶޔௗ׎ԋǶ

1.5 දҥܳೈқϐᄬ׎ᙯᡂ

ࣁΑ຾΋؁ᕕှදҥܳೈқޑᙯඤᐒڋǴ೚ӭࣴزаϯᏢӝԋБԄ܈ࢂख़ಔ מೌౢғදҥܳᴏ两բࣁൂᡏϩηǴа׎ԋᜪᐘણᠼᆢǴҞޑӧ௖૸දҥܳೈқ বঁ୔ୱჹᄬ׎ᙯᡂаϷགࢉૈΚϐख़ा܄ǺӃ߻ࣴزᡉҢǴ౽ନදҥܳೈқữ

୷ ᆄ ׇ ӈ 23-88 ϐТ ࢤࡕ Ǵ٠ ό ཮ቹ ៜ஻ Ԗ scrapie ϐλႵઓ࿶҆ಒझዦ N2a ȐneuroblastomaǴScN2aȑϐPrP^Sc׎ԋ⁴⁶ǹԜѦǴऩࢂஒλႵϐPrP୷ӢকନǴϐ ࡕᙯࢉΕȐtransfectȑ౽ѐPrPữ୷ᆄ49ঁữ୷ለϐPrPँᡂ୷ӢǴԜ୷Ӣᙯ෗λ Ⴕϝё೏scrapieགࢉǹΞǴPrP౽ନ23-88ᆶ141-176ٿТࢤϐᄒอೈқPrP106 Ȑtruncated PrP106ȑૈӧ୷Ӣᙯ෗λႵύ׎ԋᜪ՟PrP^Scϐ่ᄬ⁴⁷ǹӧN2aಒझύǴ ऩPrPલЮ114-121Тࢤ߾คݤᙯᡂԋPrP-res⁴⁸Ǵҗа΢ࣴز่݀ளޕදҥܳೈқ ᄬ׎ౢғᡂ౦ޑ୔ୱऊԾׇӈ106-147໒ۈǶ

Ԑ߻ࣴزճҔϯᏢӝԋϐදҥܳᴏ两Ҟޑࢂࣁפр೷ԋදҥܳೈқᄬ׎ว ғᙯᡂޑᜢᗖ୔ୱǶҞ߻ࣁЗᇡࣁදҥܳᴏ两Тࢤ108-144ࢂתᄽᄬ׎ᙯඤख़ा

فՅǺΓᜪදҥܳᴏ两Ȑhuman prion peptideǴhuPrPȑТࢤ106-126аϷ127-147

ૈԾว܄׎ԋڀԖᜪᐘણᠼᆢ܄፦ϐᄬ೷⁴⁹ǹΞѦу௶ճ٥ঊႵදҥܳᴏ两 Ȑsyrian hamster prion peptideǴSHaPrPȑТࢤ106-141ૈ୼׭ڋҗPrP^sen  PrP^res Ȑprotease-sensitive PrPǴPrP-senǹprotease-resistant PrPǴPrP-resȑϐᄬ׎ᙯᡂ⁵⁰Ǵ ᡉҢදҥܳТࢤ106-141ୖᆶΑPrP^CᆶPrP^Sc໔ϐբҔ⁴⁸ǹஒSHaPrP109-141ྋܭғ

౛ᐚࡋϐНྋనύ٠аFTIRϩ݋Ǵว౜ځ่ᄬҗคೕ߾סԔȐrandom coilȑᙯᡂ ࣁE-sheetǴЪ׎ԋE-sheet่ᄬࡕૈ୼ᆫ໣׎ԋᠼᆢ٠ჹೈқ䁙ڀԖל܄⁵¹ǹԶ SHaPrP109-141ᆶPrP^CҬϕբҔࡕǴૈ٬PrP^Cᕇளᜪ՟PrP^Scϐ܄፦⁵²Ǵ೭٤ࣴز่

݀ᡉҢදҥܳೈқύׇӈ106-147ёૈੋϷᄬ׎ᙯᡂਔЬाᡂ౦୔ୱǶ!

!!!!Ӄ߻ࣴزᡉҢǴϯᏢӝԋޑPrP106-126ନΑ೷ԋઓ࿶ಒझԝΫѦᗋૈߦ຾ࢃ

ރጤ፦ಒझቚғ⁴⁹ǴPrP106-126Тࢤޑữ୷ᆄڀཱུ܄ȐKTNMKHMȑǴᆙௗ๱ࢂ

ڀߚཱུ܄ȐAGAAAAGAVVGGLGȑޑ׀ЃǴځύPrP113-120ȐAGAAAAGAȑ

೏ᇡࣁࢂ׎ԋE-sheetޑख़ाТࢤ⁵³ǴฅԶ۳߻ۯ՜ԿPrP106ᚆữለȐlysineǴKȑ Տ࿼ว౜PrP106-122ӧғ౛ރᄊpHਔǴᴏ两่ᄬӭаD-helix/random coil׎ԄӸӧ

54Ǵ೷ԋԜ౜ຝޑচӢёૈࢂӢࣁ஥҅ႝޑᚆữለफ़եΑ׎ԋE-sheetਔϩη໔ޑ բҔΚǶԜѦǴBlondelle et al.මࡰрǺ൤֖ЧữለȐalanine-richȑޑᴏ两Ǵӵ KYA13KǴڀԖё׎ԋE-sheetޑૈΚ⁵⁵ǴԶவϩηኳࠠύҭᡉҢදҥܳޑᡂ׎

Ȑtransformationȑ౐ੋډAGAAAAGA୔ୱ⁵⁶ǴᆕࡴԶፕǴSHaPrP113-120ӧᜪᐘ ણᠼᆢ׎ԋၸำύתᄽख़ाفՅǶ

1.6 දҥܳϐၠނᅿ໺ࢉ

வࢬՉੰᏢفࡋᢀჸǴӧम୯ǴӢࣁᗯ१ФဂޑႩ਑рΑୢᚒԶᏤठදҥܳ

ೈқவԲဂύၠၸࡀምགࢉΑФဂǹΓᜪ߾ӢࣁӞΑғੰޑФܭࢂදҥܳೈқΞ

ၠၸΑΓᆶФ໔ޑᅿ௼ࡀምȐspecies barrierȑགࢉΑΓᜪǶTSEύ܌ࡰޑᅿ௼ࡀ ምջࢂ྽AނᅿޑTSE agentགࢉBނᅿਔǴBނᅿჹTSE੯ੰڀԖל܄Ȑresistanceȑ

57Ƕӧ೭ᜐ܌ࡰޑל܄Ȑҭᆀࡀምȑջࢂӧಃ΋жȐprimary passageȑදҥܳੰ

চҗAނᅿ໺ࢉ๏BނᅿၸำύǴ೏གࢉޑBނᅿᆶAނᅿ࣬ၨΠǴሡाၨߏ܈ࢂ

όӕޑ୻Ꭶਔ໔Ȑincubation timeȑฅࡕω཮ᄌᄌޑԝѐǴฅԶ٠ߚ܌ԖޑBނᅿ

ࣣ཮གࢉදҥܳੰচԶठੰǶӵ݀຾΋؁ӧBނᅿ໔ಃΒжȐsecond passageȑག

ࢉޑ၉ǴΨ൩ࢂBނᅿ໔ޑӕᅿ໺ࢉǴBނᅿठੰޑਔ໔཮ᕭอࡐӭȐკ1.7ȑǶԐ ය ࣴ ز Γ ᜪ ද ҥ ܳ ੯ ੰ ε ӭ а ᡫ ߏ ᜪ ୏ ނ ࣁ ჴ ᡍ ჹ ຝ Ǵ ੝ ձ ࢂ ໵ ฎ ฎ ȐchimpanzeeȑᆶႵญȐsquirrel monkeyȑࣁЬ⁵⁸Ǵલᗺࢂӧಃ΋ж໺ࢉύሡाߚ தΦޑ୻Ꭶਔ໔ԶЪܳ຦ǴӢԜׯճҔჴᡍ࠻ԴႵ຾Չ໺ࢉჴᡍǴՠว౜λႵࡐ ᜤڙΓᜪදҥܳೈқགࢉǴځ୻Ꭶਔ໔࣬྽ܭλႵғڮຼය⁵⁹Ƕ

კ 1.7 දҥܳၠᅿ໺ࢉǶȐaȑόӕޑ prion strain གࢉ࣬ӕނᅿǴ࿶ၸኧж౽ᅿ Ȑserial passagesȑࡕനࡕ཮׎ԋόӕޑठੰࠠᄊᆶੰቻȐٯӵ୻Ꭶਔ໔ᆶཞ্௃

ݩȑǴӧႝݚკ΢ё࣮ډനࡕ׎ԋόӕޑ PrP^ScǴᗨฅࣣڀԖೈқ䁙 K ל܄Ǵՠ ځελȐsizeȑࠅό࣬ӕǶȐBȑவࢌ΋ނᅿϩᚆрϐදҥܳੰচ೯தࡐᜤ໺ࢉ

๏ځдނᅿȐ୻Ꭶਔ໔ߏȑǴ೭ࢂӢࣁਈᜄޣᆶௗڙޣޑදҥܳೈқׇӈόӕԖ ᜢǴӢԜ཮ߔᛖ PrP ᄬ׎ޑᙯᡂǶฅԶӧ࿶ၸኧж౽ᅿࡕǴ୻Ꭶޑਔ໔཮ᅌᅌᕭ อǴ೭ᅿ౜ຝᆀࣁ፾ᔈ܄ȐadaptationȑǶӧࢌ٤ނᅿύǴᅿ௼ࡀምߚதଯǴջ

٬࿶ၸࡐߏޑ୻Ꭶਔ໔ϝόـԖวੰ౜ຝǴฅԶӧ೭٤ڀԖל܄ޑނᅿύஒځတ

೽๧ڗрϐදҥܳགࢉځдނᅿࠅڀགࢉΚǶऩࢂ΋໒ۈǴӧ࣬ӕޑނᅿύӧௗ

ᅿΑ࣬ӕޑѦٰޑදҥܳࠅр౜ߏਔ໔ޑ୻Ꭶਔ໔Ǵٗ൩߄Ң PRNP ޑӭࠠ܄

Ȑpolymorphismȑ೷ԋ೭ᅿ౜ຝǴ೭Ψ൩ࢂ܌ᒏޑགࢉࡀምȐtransmission barrierȑ

60Ƕ

Ȩᅿ௼ࡀምޑр౜ᆶਈᜄޣȐdonorȑϷௗڙޣȐrecipientȑޑPrPữ୷ለׇ

ӈԖᜢȩǶ཮ගр೭ঁ౛ፕࢂӢࣁКჹথ٢ᜪ୏ނ໔PrPׇӈࡕ܌ளޑ่ፕ⁶¹Ǵځ

നЬाޑ᛾ᏵࢂӢࣁว౜λႵᆶ௶ճ٥ঊႵ໔Ԗࡐଯޑᅿ௼ࡀምǺλႵচҁό཮

ڙډ௶ճ٥ঊႵPrP^ScགࢉǴՠऩஒ௶ճ٥ঊႵϐPrP୷Ӣᙯ෗ΕλႵᡏϣǴ཮٬

ᙯ෗λႵڙ௶ճ٥ঊႵPrP^Scགࢉᐒ཮εቚ⁶²ǹԜѦPrusinerஒ௶ճ٥ঊႵPrP^Scௗ ᅿΕӅԖ߄౜௶ճ٥ঊႵᆶλႵPrPϐ୷Ӣᙯ෗Ⴕᡏϣࡕว౜ǴԜ୷Ӣᙯ෗Ⴕѝ

ૈౢғ௶ճ٥ঊႵPrP^ScǴӕኬޑǴऩஒλႵPrP^ScௗᅿΕӅԖ߄౜௶ճ٥ঊႵᆶ λႵPrPϐ୷Ӣᙯ෗Ⴕᡏϣࡕว౜ǴԜ୷Ӣᙯ෗ႵѝૈౢғλႵPrP^{Sc29; 63}Ƕவག

ࢉscrapieޑλႵઓ࿶҆ಒझዦȐSc⁺-MNBȑಒझٰ௖૸දҥܳೈқᄬ׎ᙯᡂޑᜢ ᗖ ୔ ୱ ჴ ᡍ ύ ҭ ว ౜ PrP-sen ᙯ ᡂ ԋ PrP-res ਔ ੋ Ϸ ނ ᅿ ޑ ஑ ΋ ܄ Ȑ species specificityȑǺӧSc⁺-MNBಒझύ߄౜ँᡂȐஒλႵPrP108ᆶ111Տ࿼ँᡂࣁMetȑ

܈ࢂख़ಔλႵPrPȐԜλႵ஥Ԗ௶ճ٥ঊႵPrPϐM111ȑ཮υᘋλႵϣғ܄PrP-sen ᙯᡂࣁPrP-resǴ೭߄ҢӧPrP-resᆶPrP-sen໔ޑ࣬৒܄ȐcompatibilityȑᆶբҔΚ ჹܭPrP-resޑ׎ԋаϷTSE੯ੰޑ໺ኞתᄽख़ाفՅ⁶⁴Ƕ

1.6.1 λႵᆶ௶ճ٥ঊႵ໔ޑᅿ௼ࡀም

ᗨฅǴ௶ճ٥ঊႵᆶλႵΒޣ໔දҥܳೈқữ୷ለׇӈԖ94ʘޑ࣬՟ࡋȐѝ Ԗ16ঁữ୷ለόӕȑǴՠவ΢ॊ่݀ךॺளޕǴ௶ճ٥ঊႵᆶλႵ໔ӧදҥܳ੯

ੰ໺ࢉ΢Ӹӧᅿ௼ࡀምǴӢԜλႵόܰགࢉ௶ճ٥ঊႵPrP^ScǹฅԶவᙯ෗λႵ ᆶಒझ୻Ꭶჴᡍύว౜ǴՏܭPrPೈқޑύ໔୔ୱȐPrP112-187ȑځữ୷ለׇӈ

࣬՟ำࡋჹܭPrP-senᙯᡂࣁPrP-resࢂख़ाޑ ^{64; 65; 66}Ƕ

ӧલЮࢲᡏϣȐin vivoȑදҥܳ੯ੰ໺ࢉޑჴᡍ௃ݩΠǴճҔࢲᡏѦȐcell-free systemȑჴᡍٰᢀჸTSE agentӧόӕނᅿ໔ჹܭᅿ௼ࡀምགࢉޑ௃׎^{67; 68}Ƕࢲᡏ Ѧჴᡍ᛾ჴǴऩࢂஒ҅தޑPrP^CӃکςӸӧޑPrP^Sc୻Ꭶӧ΋ଆǴ཮ߦ٬PrP^Cᙯᡂ ԋPrP^ScǶ೭໨่݀ό໻᛾ჴදҥܳ੯ੰޑጭۯȐpropagationȑӸӧܭPrP^CᆶPrP^Sc ϐ໔Ǵ׳ӆԛᡍ᛾ൂ΋ೈқ౛ፕ⁵⁸ǶKocisko฻Γϩձஒಒझ߄ၲࡕаխࣝ؈ᐘݤ ԏ໣ޑ³⁵S኱ᇞޑλႵϷ௶ճ٥ঊႵϐPrP^CᆶλႵϷ௶ճ٥ঊႵϐPrP^ScܭࢲᡏѦ

຾Չ୻ᎦǴว౜λႵޑPrP^Cѝૈ೏λႵޑPrP^ScᇨᏤ೷ԋ่ᄬׯᡂǴฅԶ௶ճ٥ঊ ႵޑPrP^CǴٿނᅿϐPrP^Scࣣёჹځ೷ԋቹៜȐॶளݙཀޑࢂբޣӢࣁа3F4ൂਲ਼ לᡏ຾Չխࣝ؈ᐘǴӢԜԴႵPrP^CޑL109HaᆶV112Ha೏ׯࣁᆶ௶ճ٥ঊႵ࣬ӕޑ MetǴӃ߻ךॺჴᡍ࠻ࣴزаදҥܳᴏ两຾ՉλႵᆶ௶ճ٥ঊႵЇ඲ჴᡍޑК

ၨǴ่݀ว౜྽139Տ࿼ࣁMetਔǴ109ᆶ112Տ࿼ޑׇӈ࣬՟ࡋჹЇ඲ਏ౗ࡐख़

ा⁶⁹ȑǶKocisko฻Γ٠଺Α೚ӭόӕߏࡋޑදҥܳೈқǴ่݀ว౜ӧ138Mo/139Haǵ 154_Mo/155_HaаϷ169Mo/170_HaՏ࿼ϐữ୷ለᅿᜪ཮ቹៜλႵᆶ௶ճ٥ঊႵޑၠᅿ གࢉ⁷⁰ǶԜѦӧλႵઓ࿶҆ಒझዦޑჴᡍύ׳຾΋؁᛾ܴǴൂ΃ữ୷ለޑόӕ ȐMet138Moȑ൩ૈ׭ڋλႵPrP-resޑ׎ԋȐբޣӧSC+

-MNBಒझύ߄౜chimeric mouse-hamster PrPǴӃפрჹPrP-senᙯᡂԋPrP-resޑख़ा୔ୱӧܭPrP112-187Ǵ ԶӧԜϐ໔λႵᆶ௶ճ٥ঊႵѝԖ3ঁữ୷ለόӕǴӆճҔँᡂޑБݤפр྽

I138Mਔ཮٬PrP-senคݤᙯᡂԋPrP-resȑ⁶⁴Ƕޑዴӧࢲᡏჴᡍύ᛾ჴǴٿނᅿ ໔ѝाൂ΃ữ୷ለόӕ൩ૈቹៜදҥܳ੯ੰޑ໺ࢉਏ౗ǴᖐٯٰᇥǺФޑPrP^C ӧữ୷ለ142Տ࿼ࢂIleޑ၉൩ࡐᜤஒBSE໺ࢉ๏௶ճ٥ঊႵȐ࣬྽ܭữ୷ለ139 Տ࿼ޑMetȑǹԲޑPrP^Cӧữ୷ለ142Տ࿼ऩࢂIleҭࡐᜤஒscrapie໺ࢉ๏ξԲȐữ

୷ለ142Տ࿼ࢂMetȑ^{71; 72}Ƕදҥܳೈқϐữ୷ለ138Տ࿼ϐ܌аख़ाࢂӢࣁځՏ ӧPrP-sen119-140ϐύǴёૈࢂکPrP-resወӧޑ่ӝՏ࿼Ȑbinding siteȑ⁷³ǹӧPrP^C ύǴữ୷ለ138Տ࿼ࢂ֤ပܭE-strandȐữ୷ለ128-131ȑᆶD-helixȐữ୷ለ144-154ȑ ύޑᕉރȐloopȑ่ᄬϣǴӢԜᎃ߈PrP^C܈PrP^ScՏ࿼ޑữ୷ለࣣԖёૈࢂPrP^Cᙯ ඤԋPrP^Sc؁ᡯޑख़ाቹៜᜢᗖ⁶³Ƕ

வᙯ෗λႵჴᡍǵԲཝ᝚ੱགࢉޑಒझ୻Ꭶჴᡍǵ΋ޔډࢲᡏѦჴᡍࣣ᛾

ჴǴPrPׇӈޑ΋ठ܄ჹදҥܳ໺ࢉਏ౗תᄽख़ाفՅǴฅԶჹܭPrPữ୷ለׇӈ ӵՖ௓ڋᅿ௼ࡀምޑᐒڋϝฅόమཱǴ߈ԃٰǴςૈӧ၂ᆅύ຾ՉදҥܳޑЇ඲

ȐseedingȑჴᡍǴӧԜჴᡍύճҔख़ಔΓᜪǵԴႵаϷ௶ճ٥ঊႵޑPrP23-144

຾ՉЇ඲Ǵว౜ữ୷ለ138ӧаނᅿࣁ٩ӸȐspecies-dependentȑޑЇ඲஑΋܄ჴ ᡍύתᄽख़ाفՅǴӕਔҭ᛾ܴӧόӕނᅿ໔ޑҬᒱЇ඲ࡀምȐcross-seeding

barrierȑ཮Ӣࣁ೴ᅌޑ፾ᔈȐadaptationȑԶ೏۹ౣǴ٠Ъ཮ౢғཥޑǵόӕΒભ

่ᄬᆶόӕᜪᐘણᠼᆢࠠᄊϐᜪᐘણstrainǶฅԶӧԜჴᡍύλႵᆶ௶ճ٥ঊႵ PrP23-144໔ޑགࢉࡀምჴᡍᆶࢲᡏϣޑჴᡍ٠όֹӄ΋ठ⁷⁴ǺճҔεဉఎ๵߄౜

ख़ಔλႵϐMoPrP23-144ᆶ௶ճ٥ঊႵϐShaPrP23-144Ǵว౜ς׎ԋᜪᐘણᠼᆢ ϐλႵPrP23-144Ȑ2% w/wȑ٠คݤ٬௶ճ٥ঊႵPrP23-144ᕭอғߏᒨᅉයȐlag phaseȑǴฅԶς׎ԋᜪᐘણᠼᆢϐ௶ճ٥ঊႵPrP23-144Ȑ2% w/wȑჹλႵ PrP23-144຾ՉЇ඲ਔࠅૈᕭอځғߏᒨᅉය^{74; 75; 76}Ƕ

1.6.2 ΓᜪᆶФϐ໔ޑᅿ௼ࡀም

म୯ئФੱޑᛈวᡣךॺ࣬ߞBSEޑPrP^Scёаၠຫᅿ௼ࡀም໺ኞ๏ΓᜪǴ೭

ࢂց߄Ң೸ၸBSEޑPrP^Scૈ٬ΓᜪPrP^CᙯᡂԋPrP^Sc?ϐ߻මԖჴᡍᡉҢऩࢂע BSEޑPrP^ScௗᅿԿ஥ԖΓᜪPrP^Cϐ୷Ӣᙯ෗Ⴕคݤ٬Ԝ୷Ӣᙯ෗Ⴕวੰ⁷⁷Ƕฅ ԶǴࢲᡏѦჴᡍ᛾ჴФPrP-resૈ٬ΓᜪPrP-senᙯᡂࣁΓᜪPrP-res ^{59; 68}ǶԜѦǴё а߄౜ФPrP^C୷ӢલѨλႵ (PRNP^0/0)৒ܰڙډBSEགࢉ٠Ъ߄౜рکФགࢉ

BSEޑӕኬੰቻ⁷⁸Ǵ࣬ӕޑᙯ෗λႵӕኬ৒ܰڙډΓᜪvCJDаϷscrapieགࢉǺڙ ډΓᜪvCJDགࢉޑᙯ෗λႵځወҷයȐऊ250ϺѰѓȑǵઓ࿶ੰ౛аϷPrP^Scޑࠠ

ᄊᆶڙډФBSEགࢉޑᙯ෗λႵߚதᜪ՟ǴҗԜ௢ෳЇวΓᜪvCJDکФBSE ޑ གࢉނ፦ᜪ՟^{68; 72}Ƕ

ӧ Γ ᜪ ද ҥ ܳ ୷ Ӣ ஏ ዸ η 129 Ȑ PRNP code 129 ȑ Տ ࿼ Ӹ ӧ Ҙ ౷ ữ ለ ȐmethionineǴMetȑᆶ㐧૛਽ለȐvalineǴValȑӭࠠ܄Ȑpolymorphismȑ⁷¹ǶԿ Ҟ߻ࣁЗǴԖѤᅿΓᜪPrP^Sc೏ᇡࣁᆶόӕޑCJD߄౜ࠠԖᜢǺڂࠠޑȐଽว܄ᆶ གࢉ܄ȑCJDੰ஻஥ԖPrP^Sc1-3ࠠǴԶvCJDੰ஻߾ࢂӸӧPrP^Scಃ4ࠠǹಃ1ǵ4ࠠ

ޑPrP^ScѝӸӧΓᜪMet¹²⁹ӕ่ࠠӝηȐhomozygoteȑǴԶಃ3ࠠPrP^ScӸӧԿϿ֖Ԗ

΋ঁVal¹²⁹ჹଽ୷ӢȐalleleȑǴԶಃ2ࠠޑPrP^Scᆶஏዸη 129คᜢ⁷⁹ǴӢԜΓᜪ PrP129ޑӭࠠ܄ޑ੝፦཮ቹៜΓᜪჹදҥܳ੯ੰޑ௵གࡋЪ஻ޣև౜ᄒฅόӕ

ޑठੰ߄ࠠ⁸⁰Ƕ

ൂ΃ữ୷ለޑׯᡂӧόӕނᅿ໔ό໻کགࢉࡀምԖᜢǴӧ࣬ӕޑނᅿϣҭᆶ

ࢂց৒ܰڙډTSE੯ੰགࢉ࣬ᜢǶ಍ीၗ਑ᡉҢǴ஥Ԗஏዸη129ᙯ᝿ԋMet/Met

܈ ࢂ Val/Val ӕ ࠠ ่ ӝ η ޑ ୷ Ӣ ࠠ ᑡ ஻ ଽ ว ࠠ CJD ޑ ᐒ ౗ ၨ ౦ ࠠ ่ ӝ η Ȑheterozygousȑޑ୷Ӣࠠޑᐒ౗ଯ⁸¹ǶฅԶჹvCJDੰ஻ٰᇥǴځ୷ӢࠠѝӸӧ Met/MetࠠԄǹ࣬ჹܭ129MMǴऩ129Տ࿼р౜ޑࢂM/V܈ࢂV/VǴၨόܰᑡ஻

vCJD٠ёૈڀԖၨߏޑወҷය⁸²Ƕ

ᗨฅ129Տ࿼ޑӭࠠ܄،ۓࢂց৒ܰڙډදҥܳ੯ੰགࢉǴฅԶவғނނ౛Ꮲف ࡋٰ࣮Ǵჹܭख़ಔPrP^C129Տ࿼ϐMetᙯᡂԋValਔǴځ୏ΚᏢȐkineticȑǵᄙ᠄

ȐfoldȑаϷ዗୏ΚᏢᛙۓࡋȐthermokinetic stabilityȑࣣคख़ाቹៜ^{68; 76; 83}Ƕ೭ ߄ҢׯᡂදҥܳޑጭۯȐpropagationȑ٠όࢂ೸ၸ129M/Vӭࠠ܄ჹPrP^CޑቹៜǴ Զࢂ೸ၸჹPrP^Scޑ่ᄬ܈ࢂჹᜪᐘણᠼᆢ׎ԋ୏ΚᏢޑቹៜǶ

҅தPrP^CᙯᡂࣁPrP^ScǴӧӕᅿόӕ೽Տǵ܈όӕᅿ୏ނёૈౢғόӕϐቹ ៜǴӢԶᡣεৎख़ཥࡘԵᅿ௼ࡀምޑୢᚒǶӢࣁᅿ௼ࡀምёаှញࣁՖ΋ᅿ୏ނ ޑ౦தPrPၨᜤЇଆќ΋ᅿس୏ނϐදҥܳ੯ੰǶᆕӝа΢܌ॊǴᅿ௼ࡀምᆶPrP ϐữ୷ለׇӈԖᜢǴѦٰགࢉྍϐPrP^Scữ୷ለׇӈຫႽஎЬϐPrP^Cữ୷ለׇӈ ޣǴຫܰளډදҥܳ੯ੰǴϸϐ߾ၨᜤǶ೭ΨှញΑࣁՖԲКၨ৒ܰ໺ࢉФȐٿ ޣữ୷ለׇӈ໻7ೀόӕȑǴФा໺ࢉ๏Γ߾ᜤளӭȐٿޣữ୷ለׇӈԖ22ೀό ӕȑΖ

1.6.3 Γᜪᆶജϐ໔ޑᅿ௼ࡀም

ኧԭ࿤ۚՐӧчऍޑᘪΓ཮ӧѺᘪࡕ१ҔജȐdeerȑᆶᗸജȐelkȑǴԶ೭٤ ജԺύς೏᛾ჴ཮ӸԖCWD prion⁸⁴ǴӢԜךॺᚶᅪCWDࢂց཮ႽئФੱ΋ኬǴ

ၠނᅿ໺ࢉ๏Γᜪǻډ౜ӧךॺ٠όమཱΓᜪࢂց཮གࢉCWD܈ཥࠠᄊவ୏ނ ԶٰޑTSE੯ੰǴՠၨགݒኃޑࢂډҞ߻ࣁЗම࿶ӧᛈวεೕኳCWDޑ୔ୱȐऍ

୯ࣽᛥ܎ӭԀᆶ࠶ཥநԀȑ٠҂ว౜ΓᜪԖᜪ՟஻ੰޑ௃ݩวғ⁸⁵ǶӃ߻୷ܭғ ނӼӄԵໆǴჹܭᅪ՟ᑡ஻CJDޑੰΓคݤ຾ՉှওዴᇡǴӢԜคݤᘐۓ೭٤Ӣ

ੰԝΫޑ஻ޣࢂց੿ޑࢂӢࣁ१ҔജԺԶགࢉΓᜪTSEǴฅԶᒿ๱ਔ໔຾৖Ǵჹ ܭѳਔಞᄍ१ҔജԺ٠Ъᑡ஻CJDޑ஻ޣӧځԝࡕ຾Չှও٠҂ว౜ԖҺՖό

҅த܈ࢂཥࠠᄊදҥܳೈқޑӸӧ^{86; 87}Ǵځд໔ௗ᛾ᏵҭᡉҢΓᜪགࢉCWDޑ॥

ᓀࢂեޑǺRaymond et al.ࡰрǴCWDૈ٬Γᜪख़ಔPrPᙯᡂԋᜪᐘણᠼᆢޑਏ౗

٠όଯǴӵӕBSEᆶscrapie΋ኬǴӢࣁϩηޑό࣬৒܄ȐincompatibilityȑǴ٬ள ΓᜪPrP^CӧၠނᅿȐcross speciesȑགࢉਔόܰᙯᡂԋPrP^Sc67Ƕ

ࣁΑှCWDᆶCJD஻ޣځੰ౛ಔᙃаϷPrP^Scޑғϯ੝܄ࢂց࣬՟ǴӢԜXie et al.Кၨஏዸη129ࣁӕ่ࠠӝηȐMet/MetȑޑvCJDੰ஻ᆶགࢉCWDޑജᆶᗸ ജ⁸⁸Ǵว౜คፕࢂவੰ౛ಔᙃ܈ࢂװᔐҞ኱Ȑtarget organȑᗋࢂᗐϯޑࠠԄ Ȑglycoform patternȑǴprion strainӧၠނᅿགࢉਔ٠ό཮ߥ੮চҁޑࠠᄊ89; 90; 91;

92ǶԶࣁΑ຾΋؁ࣴزᗸജᆶΓᜪȐelk-humanȑϐᅿ௼ࡀምǴKong et al.ஒᗸജ CWDȐelk CWDȑௗᅿԿёа߄౜ΓᜪPrP܈ࢂelk PrPϐᙯ෗λႵǴёа߄౜elk PrPϐᙯ෗λႵӧௗᅿ118-142Ϻࡕ཮วੰǴฅԶ߄౜ΓᜪPrPȐ129Mȑϐᙯ෗λ Ⴕ ޔ ډ 657 Ϻ ܈ ࢂ ׳ ߏ ޑ 756 Ϻ Ǵ ࣣ ҂ ว ৖ р Һ Ֆ TSE ޑ ੰ ቻ⁹³Ǵ Ԝ ่ ݀ ᆶ Tamgüney et al.ࣴز΋ठǴёа߄౜ΓᜪPrPޑᙯ෗λႵ٠ό৒ܰགࢉவчऍεف ജȐmule deerȑ܈ࢂқ׀ജȐwhite-tail deerȑҭ܈ࢂᗸജȐelkȑԶٰޑCWD⁹⁴Ƕ ᗨฅ໔ௗ᛾Ᏽ᛾ჴΓᜪᆶജϐ໔ޑᅿ௼ࡀምࡐଯǴCWDࡐᜤགࢉΓᜪǴՠ මԖൔᏤࡰрᡫߏᜪ୏ނࠅ৒ܰགࢉCWDǴ࿶җintracerebral inoculationޑႵญ Ȑsquirrel monkeyȑ཮೏CWDགࢉ⁹⁵ǴฅԶӧᡫߏᜪ୏ނύǴཥШࣚญȐnew world monkeyȑځPRNP୷ӢׇӈکΓᜪ࣬ৡࣗӭӢԜคݤ᛾ჴΓᜪදҥܳೈқёа೏

CWDϐPrP^Scᙯඤ⁹⁶Ƕ

1.7 ځдނᅿϐᅿ௼ࡀምޑࣴز

໺ኞ܄ພတੰȐTransmissible mink encephalopathyǴTMEȑᆶ㵦ੇᆟރတੰ

ȐFeline spongiform encephalopathy FSEȑҭࢂၠຫᅿ௼ࡀም܌ᏤठޑTSE੯ੰǴ TMEޑวғࢂӢࣁឪ१೏scrapieԦࢉ܈BSEԦࢉޑԺԖᜢ⁹⁷ǴԶFSEޑวғ߾ࢂ

ӞΑᎁBSEགࢉޑᚲނႩ਑࣬ᜢ⁹⁸Ǵ྽ᛈว೭٤୏ނޑ໺ࢉ܄ੇᆟރတੰਔǴך ॺό࿣ᏼЈࢂցᗋԖځдӞΑ࣬ӕᚲނႩ਑܈ࢂԺᇙࠔޑ୏ނҭԖёૈᑡ஻

TSE੯ੰǴԶ೭٤஻ੰޑނᅿࢂց཮ԋࣁགࢉΓᜪޑύᝩޣǶฅԶவӃ߻ࣴزᡉ ҢټηȐrabbitȑǵଭȐhorseȑǵ፜ȐpigȑᆶކȐdogȑ೭٤ৎ੬ϷᚲނǴឦܭ ό৒ܰ೏දҥܳགࢉ⁹⁹ޑ୏ނǶࣁՖټηǵଭǵ፜ᆶކჹܭදҥܳགࢉڀԖל܄ǻ ӧPrPữ୷ለׇӈύǴΓॺനགᑫ፪ޑՏ࿼ՏܭPrP165-175೭ࢤ୔ୱǴখӳࢂಃ

ΒঁE-strainᆶಃΒঁD-helixౢғloopȐE2-D2 loopȑޑӦБǴᗨฅথ٢ᜪ୏ނޑ PrPữ୷ለׇӈԖଯၲ85%-95%ޑ࣬՟ࡋǴฅԶ྽PrPữ୷ለวғׯᡂਔ൩཮٬᏾

ᡏ่ᄬׯᡂǴᏤठPrP-resόܰౢғӢԜගଯᅿ௼ࡀምǶE2-D2loopޑՏ࿼ёૈࢂ

کprotein XౢғբҔޑӦБǴԶprotein X౒ෳࢂୖᆶPrP^CᙯᡂࣁPrP^ScޑᇶӢη Ȑco-factorȑ¹⁰⁰ǴӢԜǴऩE2-D2loop่ᄬၨࣁڰۓȐrigidityȑޑ၉Ǵ൩Ԗёૈ཮

ቚуނᅿڙPrPགࢉޑᐒ཮¹⁰¹ǹټηᆶ፜ӧPrP173Տ࿼ࣣࣁSerǵଭϐPrP167Տ࿼

ࣁSerǵԶކӧPrP177Տ࿼ࢂArgǴ೭٤ữ୷ለନΑ཮کځᎃ߈ޑữ୷ለȐӵǺټ ηکކϐAsp177-Arg163ȑౢғᡶᐏȐsalt bridgesȑǵణᗖȐhydrogen bondsȑ܈

ࢂ౧Н܄ޑௗ᝻Ȑhydrophobic contactȑѦǴữ୷ለҁيޑғނނ౛੝܄ҭ཮ቹ ៜE2-D2loopޑ่ᄬǴӢԜټηǵଭǵ፜ᆶކό৒ܰڙදҥܳགࢉ 102; 103; 104

Ƕ!

1.8 ځдදҥܳᅿ௼ࡀምޑ࣬ᜢࣴز

ೈқᒱᇤᄙ᠄ൻᕉܫεȐProtein misfolding cyclic amplification, PMCAȑǴ΋

ᅿЇҔPCRཷۺ܌ࣴวрޑཥמೌǴࢂӧ2001ԃҗClaudio SotoวܴǴPMCAૈӧ in vitroޑ௃ݩΠኳᔕPrP^ScޑፄᇙၸำǴջճҔϿໆޑPrP^ScᇨᏤhostޑPrP^Cᄬ׎ᙯ

ᡂǴ٠ౢғཥޑεໆPrP^Sc105ǺSotoஒscrapie-infectޑঊႵတಔᙃనӃаPKೀ౛ࡕ ӆีញډ൳ЯаՋБᗺᏀȐwestern blotȑΨคݤୀෳޑໆǴฅࡕӆஒԜঊႵတಔ ᙃనᆶ҅தঊႵတಔᙃȐPrP^Cȑ୻Ꭶӧ΋ଆǴ࿶ၸ΋ࢤਔ໔ࡕ཮ౢғཥޑPrP^Sc-like proteinǴϩηໆऊ27-30 kDaȐPrP-resȑǴԜਔӆаຬॣݢ᎜࿗ሺȐsonicatorȑஒ షӝނ᎜࿗Ǵฅࡕว౜PrP-res཮ᗊቚǶӧԜ௃ݩΠȐin vitroȑፄᇙޑprionڀԖprion ޑख़ा੝܄ǴٯӵϝԖགࢉΚ٠ЪߥԖstrainޑ஑΋܄ǶԜѦPMCA׳ૈࡷᏯԖᜢ දҥܳ੯ੰނᅿࡀምޑୢᚒǴӢࣁԜ໨מೌёаமॐPrP^CᙯᡂԋPrP^ScǴӢԜѝा

ғ ނ ᡏ ϣ Ԗ ཱུ ༾ ໆ ޑ PrP^ScӸ ӧ ൩ ૈ ᙖ Ԝ מ ೌ Ԗ ਏ ޑ ፄ ᇙ Ӛ ᅿ ό ӕ ޑ prion strains/speciesǴჹܭ΋٤ڀprion-resistantޑނᅿ܈೚ૈᙖҗԜБݤѺઇǴԶЪΨ

ૈమཱޕၰ೭٤ڀԖprion-resistantޑނᅿࢂց཮ԋࣁ٬Γᜪགࢉදҥܳ੯ੰޑ ύᝩޣ ¹⁰⁶ǶVidal E฻Γමஒټηᆶކޑတ֡፦నᆶBSEޑੰတ֡፦న຾Չ PMCAࣴزǴ่݀ว౜BSEޑੰတ֡፦నύޑPrP^Scሡा࿶ၸԿϿΎԛPMCAϸᔈ ωё٬ټηᆶކޑ҅தတ֡፦నύޑPrP^CౢғཥޑPrP^ScǴஒԜཥޑPrP^ScௗᅿԿ ёа߄౜Γᜪ܈ࢂФPrP^Cϐ୷Ӣᙯ෗λႵࡕว౜ǴԜཥޑPrP^ScϝߥԖBSE prion ޑ੝܄คݤ٬୷Ӣᙯ෗ႵวੰǴӢԜჹټηᆶކٰᇥऩࢂ΋໒ۈڙprionགࢉޑ

ٰྍٰԾBSEǴٗሶ೭٤୏ނϝࢂคݤԋࣁBSEགࢉΓᜪޑύᝩޣ¹⁰⁷Ƕ

1.9 ҁፕЎ୏ᐒ

ೈқ፦ޑ΋ભ่ᄬ،ۓځΟભ่ᄬǴฅԶჹදҥܳೈқٰᇥǴځữ୷ለׇӈ ჹ᏾ᡏޑೈқ፦่ᄬٰᇥ൳Яؒϙሶख़ाቹៜǴځቹៜޑࢂPrP^CᙯᡂԋPrP^Scਔޑ ᙯඤၸำ75; 76; 108

Ƕӧٿঁόӕނᅿϐ໔Ǵځදҥܳೈқ΢ѝाԖ΋ঁữ୷ለό ӕǴ൩཮ౢғᅿ௼ࡀም^{109; 110}ǶᖐٯٰᇥǴऩࢂஒλႵઓ࿶҆ಒझዦPrP΢ޑ΋ঁ

ữ୷ለ࿼ඤԋӧټηPrP΢࣬྽Տ࿼ޑữ୷ለȐӵ:N173SȑǴ߾λႵPrP^Cӧᙯ ඤԋPrP^Scၸำύ཮Ԗܢל܄¹¹¹ǶӢԜǴൂ΋ữ୷ለޑቹៜӧᅿ௼ࡀምаϷࢂց

৒ܰڙදҥܳ੯ੰགࢉ΢՟Яࢂ࣬ӕޑᐒڋǶ

ҁࣴزޑҞޑࢂаϯᏢӝԋБԄǴӝԋኧచදҥܳᴏ两ǴவϩηቫԛаϷȨໆ ϯȩᢀۺ௖૸දҥܳೈқׇӈᆶགࢉࡀም໔ޑᜢ߯Ƕךॺᄒڗදҥܳೈқදၹᇡ ࣁനख़ाޑ΋ࢤữ୷ለׇӈ108-144ࣁኳࠠ຾ՉჴᡍǶ೭٤ᴏ两ނᅿхࡴΑჴᡍ

࠻தـޑλႵǵᒲႵаϷ௶ճ٥ঊႵǴаϷම࿶ൔᏤၸёа೏BSEགࢉޑᒗکྰ

ηǴᗋԖჹදҥܳ੯ੰԖל܄ޑކᆶ፜ǴനࡕᗋԖФǵജаϷΓᜪǶ೭٤ނᅿӧ PrP108-144୔໔Ǵϩձܭ109ǵ112ǵ129ǵ135ǵ138ǵ139аϷ143฻Տ࿼ϐữ୷ ለූ୷Ԗ܌όӕǴԶ೭٤Տ࿼ӧӃ߻ࣴزύவPrP^CᙯᡂࣁPrP^Scਔתᄽख़ाفՅǴ ӢԜךॺճҔჴᡍ࠻ςวౢрޑس಍ǴவԋਡᆫӝኳࠠޑཷۺрวǴ௖૸දҥܳ

ữ୷ለׇӈӵՖቹៜᜪᐘણ׎ԋ੝܄ᆶЇ඲ਏ౗Ƕ

ಃΒകʳ ׷਑ᆶБݤ

2.1 ׷਑

2.1.1 Н

2.1.2 ϯᏢᛰࠔ

ᛰࠔ! ٰྍ!

Acetic acid, glacial J. T. Baker

Acetonitrile J. T. Baker

Dichloromethane (DCM) Mallinckrodt N,N-Dimethylformamide (DMF) Mallinckrodt Dimethyl sulphoxide, sterile filtered Sigma Dulbecco’s Modified Eagle Medium (DMEM) Gibco High Modified Dulbecco’s Modified Eagle’s Medium

(1X)(With phenol red)

Hyclone

High Modified Dulbecco’s Modified Eagle’s Medium (1X)(Without phenol red)

Hyclone

Ethanol Riedel-de Haën

Fmoc-Ala-OH AnaSpec Inc.

Fmoc-Arg (Pbf)-OH AnaSpec Inc.

Fmoc-Asn (Trt)-OH AnaSpec Inc.

Fmoc-Asp (OtBu)-OH AnaSpec Inc.

Fmoc-Gly-OH AnaSpec Inc.

26

Fmoc-His(Trt)-OH AnaSpec Inc.

Fmoc-Leu-OH AnaSpec Inc.

Fmoc-Lys (Boc)-OH AnaSpec Inc.

Fmoc-Met-OH AnaSpec Inc.

Fmoc-Phe-OH AnaSpec Inc.

Fmoc-Pro-OH AnaSpec Inc.

Fmoc-Ser (tBu)-OH AnaSpec Inc.

Fmoc-Tyr (tBu)-OH AnaSpec Inc.

Fmoc-Val-OH AnaSpec Inc.

Fmoc-Ile-OH AnaSpec Inc.

Fetal Bovine Serum (FBS) Biochemical Industries Hydrochloric acid 37% Riedel-de Haën

2-mercaptoethanol Sigma

Methanol Mallinckrodt Methyl tert-Butyl Ether (MTBE) TEDIA

N-Methylmorpholine (NMM) Acros MTT power (Thiazoly blue Tetra-zolium Bromide) Sigma

Piperidine Merck 2-propanol J.T.Baker

PyBOp Novabiochem Rink Amide AM resin Novabiochem

Sodium Acetate anhydrous Merck Sodium Chloride Merck

Sodium phosphate monobasic Riedel-de Haën Sodium phosphate dibasic dihydrate Riedel-de Haën

Tris Hydrochloric acid 99.9% (TFA) Ampesco Triisopropylsilane (TIS) Acros Tris (hydrochloride) aminomethane (Tris) Amresco Trypsin-EDTA solution (1X) Sigma

2.2 ሺᏔ

2.2.1 પϯǵϩ݋Ҕᆅࢊ

(1) ଍࣬-ଯਏన࣬ቫ݋ȐRP-HPLCȑǺVydacC18 column ( 218TP1010, 25 cm Ø 10 mm).

(2) ዗ΚᏢྋှࡋȐThermodynamic solubilityȑϩ݋ǺAgilent ZORBAX Eclipse column, 5 Pm, 4.6 mm Ø!150 mm

2.2.2 εࠠሺᏔ

(1) ғނӼӄᏹբѠǺNUAIRE biological safety cabinet AU-425-600 (2) ᇃ਻ྐ๵ଠǺTOMY SX-700 high-pressure sterilizer

(3) ᚆЈᐒǺBECKMAN COULTER Allegra^TM X-12R centrifuge (4) ༾ໆᚆЈᐒǺEppendorf centrifuge 5415D

(5) ଯਏన࣬ቫ݋ሺǺAgilent 1100 series variable wavelength HPLCȐG1379A degasser, G1311A QarPump, G1314A VWD UV detectorȑ (6) ዗ Κ Ꮲ ྋ ှ ࡋ ϩ ݋ س ಍ Ǻ Agilent 1100series Ȑ G1379A degasser, G1311A

QartPump, G1313A ALS autosampler, G1316A TCC column compartmentȑ

(7) ੿ޜհএଳᔿᐚᕭሺǺHETO MAXI dry lyo

(8) ຬॣݢѺ࿗ᐒǺDr.Hielscher UP 100H ultraschallprozessor

(9) ڰ࣬ᴏ两ӝԋሺǺAutomated solid phase peptide synthesizer PS3^TM

28

(10) ๋ѦӀ-ёـӀϩӀӀ᛼ሺǺBECKMAN COULTER DU800 UV-Visible spectrophotometer

(11) ᑻӀӀ᛼ሺǺJASCO FP-750 spectrofluorometer (12) ༝ΒՅӀ᛼ሺǺJASCO J-715 spectropolarimeter

(13) ୷፦ᇶշႜ৔ಥߕෞᚆ-ਔ໔Ԅ०Չ፦᛼ሺǺMALDI micro MX™

(14) ऀ೸Ԅႝηᡉ༾᜔ǺHitachi H-7000

2.3 Бݤ

2.3.1 දҥܳᴏ两ޑӝԋǵપϯᆶ᠘ۓ

Ȑ1ȑڰ࣬ᴏ两ӝԋݤȐsolid phase peptide synthesisȑ

ҁጇࣴز܌٬Ҕޑදҥܳᴏ两Тࢤׇӈ 108-144Ȑკ 2.1ȑࢂճҔ PS3^TMԾ

୏ ᴏ 两 ӝ ԋ ሺ Ȑ Rainin Instruments ȑ ٩ Ᏽ ڰ ࣬ ᴏ 两 ӝ ԋ ݤ Ȑ9-fluorenylmethoxycarbonylȐFmocȑȑ኱ྗำׇ܌ӝԋǹӧᴏ两ӝԋၸำύǴ а 0.1 mmole resinȐRink Amide AM resinȑբࣁЍ࡭ނǴೱ่٠ڰۓ֖ 0.4 mmole

ୁ᜘ߥៈ୷ޑữ୷ለ຾Չᴏ两уߏޑӝԋϸᔈǶFmoc ӧӝԋਔ྽բữ୷ለޑ N ᆄߥៈ୷ǴҞޑࢂٛЗୁ᜘ϸᔈޑ׎ԋǹPyBOP Ҕٰࢲϯ N-D-protected ữ୷ለ

़ғނϐ C ᆄǹN, N-dimethylformamide ȐDMFȑࢂӝԋၸำύЬाޑྋᏊǴբ Ҕӧஒҗ resin Ϸ Fmoc ߥៈޑữ୷ለ़ғނѐߥៈȐdeprotectionȑǵጠӝ ȐcouplingȑϷؑࢱȐwashingȑǶѐߥៈྋనȐdeprotection reagentȑϐଛᇙࢂஒ 20 % (v/v) piperidine ྋӧ DMFǴԶࢲϯྋనȐactivation reagentȑ߾ࢂஒ 4.45 % (v/v) N-methylmorpholineȐNMMȑྋӧ DMFǶጠӝਔ໔Ȑcoupling timeȑຎ Fmoc ߥៈޑữ୷ለϐׇӈߏࡋᆶᐚࡋٰ،ۓǶ

ӝԋֹ౥ࡕǴresin ٩ׇ࿶ၸ DMFǵethanol аϷ DCM ؑࢱǴϐࡕӆஒ resin

࿼ܭ੿ޜύ 1-2 λਔ٬ځଳᔿǶ

კ 2.1 ҁࣴزύ܌٬ҔϐӚނᅿӝԋදҥܳᴏ两Тࢤׇӈ 108-144Ȑׇӈኧӷࢂ

аΓᜪදҥܳࣁ୷ྗȑǶόӕᚑՅ܌ࡰࣁаФදҥܳೈқȐbPrPȑࣁ୷ྗǴᆶځ ჹᔈϐữ୷ለූ୷ϩձࣁ 1 ঁǵ2 ঁᆶ 3 ঁϐόӕȐᒲႵǵчऍεفജǵλႵǵ ΓᜪǵঊႵϐදҥܳᴏ两ׇӈϩձڮӜࣁ bvPrPǵmdPrPǵmPrPǵhuPrP ᆶ haPrPȑǶ

ࣁΑעӝԋӳޑᴏ两வ resin ΢ϪΠȐcleavageȑǴஒ resin ܭ࠻ྕΠҔ trifluoroacetic acidΰTFAα / water / 1,2-Ethanedithiol ΰEDTα / triisopropylsilane ΰTISαа 94Κ2.5Κ2.5Κ1 ޑКٯᠳ୏ 2 λਔǶӧϸᔈၸำύǴữ୷ለޑୁ᜘

ߥៈ୷ӧԜਔ཮೏ؑࢱΠٰǴԶӧ౽ନữ୷ለୁ᜘ߥៈ୷ਔ཮ౢғ೚ӭᒃਡ܄ޑ

໚ᚆηǴԜਔሡा TISǵwater аϷ EDT ٰమନǹନԜϐѦǴEDT ᗋёҔٰ׭ڋ

࿶ၸለ໽ϯϐҘ౷ữለȐmethionineȑ܌ౢғޑ਼ϯբҔԶ೷ԋޑቹៜǶ྽܌Ԗ ϸᔈ่״ࡕǴషӝނ཮࿶җ G2 ᅅЏၸᘠǴၸᘠࡕϐనᡏޔௗуӧ 80 ml Ⴃհޑ Methyl tert-Butyl Ether ȐMTBEȑύǴG2 ᅅЏϐϾ৩ελࣁ 10~15Pm ૈஒ resin ੮ӧᅅЏǴԶᴏ两ܭ MTBE ύ؈ᐘрٰǶಉ๧ڗޑදҥܳᴏ两໪ӆ࿶ၸ 10ʚǵ ᙯೲ 4750 rpmȐऊ 3000×gȑᚆЈ 15 ϩដǴԜၸำख़ፄΟԛа౽ନ TISǵwaterǵ EDTǵTFA Ϸځдᚇ፦ǴஒᚆЈࡕϐ؈ᐘނ࿼ܭ੿ޜύ 1 λਔ٬ځଳᔿǴаഢࡕ

ុ٬ҔǶ

30

Ȑ2ȑදҥܳᴏ两ޑપϯᆶ᠘ۓ

-ଯਏన࣬ቫ݋Ȑreversed-phase high performance liquid chromatographyǴᙁᆀ RP-HPLCȑ຾ՉપϯǶ܌٬Ҕޑᆅࢊࣁ VydacC18 columnȐ218TP1010, 25 cm x 10 mm, Supelco, USAȑǶપϯၸำύǴᆅࢊ຾ՉఊࡋԄȐgradientȑؑගǴ܌٬Ҕޑ ྋనϩձࣁ Buffer AȐ֖Ԗ 94.9% water / 5% acetonitrile / 0.1% TFAȑаϷ Buffer BȐ99.9% acetonitrile / 0.1% TFA ȑǴࢬೲ೛ۓࣁ 3 ml/minǴቫ݋ၸำа UV275nm

Ϸ UV220nmϐ֎ԏॶ຾Չᅱෳೈқ፦Ƕஒ೏ؑගрϐදҥܳᴏ两ྋనԏ໣Ǵӆ࿶

җ੿ޜհএଳᔿᐚᕭሺȐHeto, Denmarkȑஒځଳᔿԋણ҃Ǵջࣁપϯֹԋޑද ҥܳᴏ两ણ҃ǴஒϐᓯӸܭ-20ʚӇጃǴаճࡕុჴᡍ٬ҔǶપϯࡕϐᴏ两ёճ Ҕ୷፦ᇶշႜ৔ಥߕෞᚆ-ਔ໔Ԅ०Չ፦᛼ϩ݋ݤȐmatrix-assisted laser desorption ionization-time-of-flight mass spectrometryǴᙁᆀ MALDI-TOF MSȑୀෳځϩηໆ ᆶપࡋǶ

Ӄஒપϯࡕޑදҥܳᴏ两ણ҃ྋӧ 1% acetic acidǵ50% acetonitrile Нྋన ύǴϐࡕӆᆶD-cyano-4-hydroxycinnamic acid (CHCA)୷፦Нྋనΰ10 mg/mL CHCA in 40 % acetonitrile and 0.1% TFAα٩฻Кٯషӝ֡ϬǴճҔ TOF ෳໆр ځϩηໆᙖа᠘ۓදҥܳᴏ两ϐيϩ٠ຑ՗પࡋǶаΠࣁ፦᛼ϩ݋Ӛӝԋϐදҥ

ܳᴏ两ϐ่݀Ǻ

Ȑaȑ bPrP

ȐbȑbvPrP

32 ȐcȑmdPrP

ȐdȑdogPrP

ȐeȑcatPrP

ȐfȑpigPrP

34 ȐgȑlionPrP

ȐhȑmPrP

ȐiȑhuPrP

ȐjȑhaPrP

კ 2.2 а MALDI-TOF MS ϩ݋ӝԋϐӚදҥܳᴏ两Тࢤ 108-144 ϐკ᛼Ƕკ᛼

ύനଯঢ়ջࣁӝԋϐӚදҥܳᴏ两ϩηໆǴځ౛གྷϩηໆࣁǺȐaȑbPrP ϩηໆ

36

ࣁ 3638.791 DaȐbȑbvPrP ϩηໆࣁ 3656.747 DaȐcȑmdPrP ϩηໆࣁ 3665.802 Da ȐdȑdogPrP ϩηໆࣁ 3620.837 DaȐeȑcatPrP ϩηໆࣁ 3670.763 DaȐfȑpigPrP ϩηໆࣁ 3611.78 DaȐgȑlionPrP ϩηໆࣁ 3697.774 DaȐhȑmPrP ϩηໆࣁ 3638.791 DaȐiȑhuPrP ϩηໆࣁ 3643.752 DaȐjȑhaPrP ϩηໆࣁ 3706.676 DaǶ

2.3.2 а༝ΒՅӀ᛼इᒵදҥܳᴏ两ϐᜪᐘણᠼᆢ׎ԋ

Ȑ1ȑදҥܳᴏ两׎ԋᜪᐘણᠼᆢϐਔ໔຾৖Ȑtime courseȑ

ࣁ Α ᢀ ჸ ࢂ ց Ӣ ࣁ ද ҥ ܳ ᴏ 两 ׇ ӈ ޑ ৡ ౦ Զ Ԗ ό ӕ ޑ ่ ᄬ ૈ ໆ ࡀ ም Ȑconformational energy barrierȑǴ٬ޑදҥܳൂᡏคݤ໩ճ຾Չᄬ׎ᙯᡂǴ຾Զ Ꮴठόӕޑགࢉਏ౗ǴӢԜᢀჸόӕނᅿϐදҥܳᴏ两Ծว܄ᜪᐘણȐamyloid fibrilȑ׎ԋၸำǶҗܭදҥܳᴏ两׎ԋᜪᐘણᠼᆢޑၸำੋϷೈқ፦Βભ่ᄬ Ȑsecondary structureȑޑׯᡂǴӢԜёа٬Ҕ༝ΒՅӀ᛼ሺȐcircular dichroismǴ CDȑٰᢀෳᜪᐘણᠼᆢޑ׎ԋ௃׎ǶஒϯᏢӝԋޑදҥܳᴏ两୻Ꭶӧᠼᆢ୻Ꭶ నਔǴ΋໒ۈ่ᄬ཮և౜คೕ߾௔ԔǴԶӧᜪᐘણᠼᆢ׎ԋਔǴ่ᄬ཮೴ᅌᙯࣁ E-structureǴԜ΋౜ຝёճҔᢀჸ 218 nm ߕ߈Ӏ᛼ૻဦǶ

200 210 220 230 240 250 -25

-20 -15 -10 -5 0 5 10 15

Ellipticity (mdeg)

wavelength(nm)

0hr 23hr 46hr 70hr 92hr 113hr 137hr 158hr 185hr 233hr

კ 2.3 ճҔ CD Ӏ᛼ᢀෳ bPrP ׎ԋᜪᐘણᠼᆢޑၸำǶදҥܳᴏ两ӧ׎ԋᜪᐘ ણၸำਔǴ่ᄬ཮җচҁޑ random coil ᙯᡂԋ -sheet ᄬ׎ǴԶ CD Ӏ᛼ૻဦΨ

཮җനեᗺޑ 200 nm ᙯ౽ډ 218 nm ޑ౜ຝǴ੝ձଞჹE-sheet ่ᄬ຾ՉᢀჸǴ ӢԜ 218 nm ߕ߈ޑૻဦҭԋࣁख़ा٩ᏵǶ

༝ΒՅӀ᛼ȐCDȑЬाࢂճҔ΋ঁѳय़ୃཱུӀȐplane polarized lightȑ೯ၸ ڀԖ௽Ӏ܄ޑނ፦ǴԶԜѳय़ୃཱུӀΞёϩှԋѓ௽༝ୃཱུӀȐright circularly polarizes lightȑϷѰ௽༝ୃཱུӀȐleft circularly polarized lightȑǴ྽Ѱѓ௽༝ୃཱུ

Ӏ೯ၸ௽Ӏ܄ނ፦ਔځ߻຾ೲࡋ٠ό཮࣬฻ӢԜ೷ԋό࣬฻ޑ֎ԏǶ༝ΒཱུӀ٩ Ᏽႝηૈ໘៌ᎂޑόӕё୔ϩԋᇻ๋ѦӀጄൎȐfar-UV , 170-250 nmȑϷ߈๋Ѧ ӀጄൎȐnear-UV , 250-350 nmȑǶᇻ๋ѦӀЬा߯Ӣೈқ፦܈ᴏ两ㄽữȐamideȑ ࣁЬाวՅიǴځݢߏጄൎឦܭ n^*Ϸ ^*Ǵё᠘ۓೈқ፦Βભ่ᄬǴӵ

-helixǵ-sheet ک random coil ฻Ǵ-helix ่ᄬޑ੝ቻࢂӧݢߏ 192 nm ߕ߈཮Ԗ

΋҅֎ԏঢ়Ȑpositive peakǴҗȐ^*ȑperpendicular transition ೷ԋȑǴԶӧݢߏ 208 nmǵ222 nm Βೀߕ߈཮Ԗॄ֎ԏঢ়Ȑnegative peakǴҗ n^* transition೷ԋȑǹ

-sheet ่ᄬ੝ቻࢂӧݢߏ 195 nm ߕ߈཮Ԗ΋҅֎ԏঢ়Ȑҗ ^* transition ೷ ԋȑǴԶӧݢߏ 218 nm ߕ߈཮Ԗ΋ቨቶޑॄ֎ԏঢ়Ȑҗ n^* transition೷ԋȑǹ

38

Կܭคೕ߾௔ԔȐrandom coilȑϐ่ᄬ੝ቻ߾ࢂӧ 195 nm ߕ߈Ԗ΋ܴᡉޑॄ֎

ԏঢ়Ƕ߈๋ѦӀ୔ϐݢߏጄൎЬाࢂᢀჸԜ୔ࢤڀԖၨம֎Ӏ߯ኧޑϩηӵữ୷ ለූ୷ύ֖ޱ३ᕉޑୁ᜘Ȑaromatic acid chainȑࣁЬǴ೯தҔܭ᠘ۓೈқ፦܈ᴏ 两ޑΟભ่ᄬȐtertiary structureȑ฻ϩηҥᡏ੝܄¹¹²Ƕ

຾Չදҥܳᴏ两Ծว܄ᜪᐘણᠼᆢ׎ԋჴᡍਔǴӃஒදҥܳᴏ两ྋܭѐᚆη Нύଛ࿼ԋ prion peptide stock solutionȐ2×ȑǴӆᆶ fibril formation bufferȐ280 mM NaCl ǵ40 mM NaOAcǴpH3.7ȑа฻КٯషӝǴளډനಖޑ prion peptide fibril formation solutiooǶҗܭදҥܳᴏ两ׇӈ֖Ԗ 1 ঁ TyrǴӢԜճҔ๋ѦӀ-ёـӀ ϩӀӀ᛼ሺӧ UV275nmϐ֎Ӏࡋ຾ՉۓໆǴ٬දҥܳᴏ两നࡕᐚࡋࣁ 0.25 mg/ml Ȑ50 PMȑǶ୻ᎦਔǴᓉ࿼ܭ 25ʚ୻ᎦጃύǴӧ؂ঁਔ໔ᗺ຾Չෳ၂߻཮Ӄа༾

ໆ֎ᆅ֎ܫྋనᠳ܏ऊ 10 ԛǴԶࡕճҔ CD ܭ؂ঁਔ໔ᗺΠᢀჸ 218nm ᡂϯޑ

௃׎Ƕෳໆਔ٬ҔޑҡमКՅኲࣁӀ৩ 1 mm ޑКՅኲȐᖼԾ Hellmaȑǹ࠻ྕΠ

௟ඔݢߏጄൎԾ 200 nm Կ 250 nmǹ࣬ᜢୖኧ೛ۓǺbandwidth ࣁ 2 nmǴscanning speedࣁ 20 nm/minǴdata spacing ࣁ 0.05 nmǶ؂ԛ؂΋ঁኬࠔӚΒᆅǴ؂ᆅ௟

ඔ 2 ԛǴ܌ளӀ᛼่݀ࣁ 2 ԛ௟ඔࡕޑѳ֡ॶǴϐࡕճҔ origin ೬ᡏ຾Չᔕӝ ȐfittingȑǴฅࡕӆीᆉрғߏᒨᅉਔ໔Ȑlag timeȑǶҗܭ؂ঁኬࠔख़ፄ 4-8 ԛό

฻ǴӢԜஒख़ፄ 4-8 ԛϐ lag time ਔ໔а bPrP ࣁ௓ڋಔ຾Չ one-way ANOVAǴ

ीᆉрӚදҥܳᴏ两ޑ lag time ᆶ bPrP ࣬КၨࢂցԖᡉ๱ৡ౦Ȑsignificant differenceȑǶϩ݋ኧᏵਔǴۘሡԌନᠼᆢ୻Ꭶྋన܌೷ԋޑङඳӀ᛼Ȑbackground spectrumȑǶ

؂΋ኬࠔϐᜪᐘણᠼᆢ׎ԋޑ lag time ीᆉǺᒿਔ໔ᡂϯϐ 218 nmȐFȑޑ ᐍ༝౗ȐellipticityǴmdegȑჹ୻Ꭶਔ໔Ȑincubation timeǴtȑϐ fitting БำԄǴ ளډ΋ߚጕ܄ǵΒԛБำԄϩ݋Ǻ